US 20040225449A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0225449 A1 Bevilacqua et al. (43) Pub. Date: Nov. 11, 2004
(54) SYSTEMS AND METHODS FOR which is a continuation-in-part of application No. CHARACTERIZING A BIOLOGICAL 09/605,581, filed on Jun. 28, 2000, now abandoned. CONDITION OR AGENT USING SELECTED GENE EXPRESSION PROFILES (60) Provisional application No. 60/141,542, filed on Jun. 28, 1999. Provisional application No. 60/195,522, (76) Inventors: Michael P. Bevilacqua, Boulder, CO filed on Apr. 7, 2000. (US); John C. Cheronis, Conifer, CO (US); Victor Tryon, Loveland, CO Publication Classification (US); Danute M. Bankaitis-Davis, Longmont, CO (US) (51) Int. Cl." ...... G06F 19/00; G01N 33/48; GO1N 33/50 Correspondence Address: (52) U.S. Cl...... 702/20 Barbara J. Carter Bromberg & Sunstein LLP ABSTRACT 125 Summer Street (57) Boston, MA 02110-1618 (US) Methods are provided for evaluating a biological condition Appl. No.: 10/781,558 of a Subject using a calibrated profile data Set derived from (21) a data Set having a plurality of members, each member being (22) Filed: Feb. 17, 2004 a quantitative measure of the amount of a Subject's RNA or protein as distinct constituents in a panel of constituents. The Related U.S. Application Data biological condition may be a naturally occurring physi ological State or may be responsive to treatment of the (63) Continuation-in-part of application No. 09/821,850, Subject with one or more agents. Calibrated profile data Sets filed on Mar. 29, 2001, now Pat. No. 6,692,916, may be used as a descriptive record for an agent. Patent Application Publication Nov. 11 9 2004 Sheet 1 of 49 ?
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SYSTEMS AND METHODS FOR criteria for Screening patients as they enter a clinical trial to CHARACTERIZING A BIOLOGICAL CONDITION ensure that the beneficial effect of a drug if it exists may be ORAGENT USING SELECTED GENE detected and quantified. EXPRESSION PROFILES SUMMARY OF THE INVENTION RELATED APPLICATIONS 0006. In a first embodiment of the invention there is 0001. This application is a continuation-in-part of U.S. provided a method, for evaluating a biological condition of application Ser. No. 09/821,850, filed Mar. 29, 2001, now a Subject, that includes: obtaining from the Subject a Sample U.S. Pat. No. 6,692,916, issued Feb. 17, 2004, which is a having at least one of RNAS and proteins, deriving from the continuation-in-part of U.S. application Ser. No. 09/605, Sample a first profile data Set, the first profile data Set 581, filed Jun. 28, 2000, which application claims priority including a plurality of members, each member being a from provisional application Ser. No. 60/141,542, filed Jun. quantitative measure of the amount of a distinct RNA or 28, 1999 and provisional application Ser. No. 60/195,522 protein constituent in a panel of constituents Selected So that filed Apr. 7, 2000. These related applications are hereby measurement of the constituents enables measurement of the incorporated herein by reference. biological condition; and producing a calibrated profile data set for the panel, wherein each member of the calibrated TECHNICAL FIELD profile data Set is a function of a corresponding member of the first profile data Set and a corresponding member of a 0002 Embodiments of the recent invention provide sys baseline profile data Set for the panel, the calibrated profile tems and methods utilizing gene expression analysis for data Set providing a measure of the biological condition of characterizing a biological condition or agent. the Subject. BACKGROUND ART 0007. In another embodiment, a method is provided for evaluating a biological condition of a Subject, that includes 0003. There has been substantial discussion including obtaining from the Subject a first Sample having at least one congressional hearings concerning medical errors. One of fluid, cells and active agents, applying the first Sample or Source of medical errors includes errors with medications. a portion thereof to a defined population of indicator cells, Upwards of 98,000 hospitalized patients annually have been obtaining from the indicator cells a Second Sample contain documented to be victims of medication errors (Statement of ing at least one of RNAS or proteins, deriving from the the American Pharmaceutical ASSociation to the Senate Second Sample a first profile data Set, the first profile data Set Appropriations Committee Labor, health and Human Ser including a plurality of members, each member being a vices Education Subcommittee Hearing on Medical Errors quantitative measure of the amount of a distinct RNA or Dec. 13, 1999). These errors include problems arising from protein constituent in a panel of constituents Selected So that drug interactions for a particular patient taking more than measurement of the constituents enables measurement of the one drug, problems concerning the response of an individual biological condition; and producing a calibrated profile data to a particular drug and incorrect medication for a particular set for the panel, wherein each member of the calibrated condition. Medical errors further arise as a result of misdi profile data Set is a function of a corresponding member of agnosis. This may occur as a result of insensitive diagnostic the first profile data Set and a corresponding member of a techniqueS or a wide range of interpersonal variability in the baseline profile data Set for the panel, the calibrated profile manner in which a clinical State is manifest. At present, there data Set providing a measure of the biological condition of are few tools available for optimizing prognosis, diagnosis the Subject. and treatment of a medical condition taking into account the 0008. In a another embodiment, a method is provided for particular phenotype and genotype of an individual. evaluating a biological condition affected by an agent, the 0004. There has been increasing interest in herbal drugs method including: obtaining, from a target population of or nutraceuticals. These compounds are grown and collected cells to which the agent has been administered, a Sample from around the World, and consequently the compounds are having at least one of RNAS and proteins, deriving from the Subject to regional and temporal differences in collection Sample a first profile data Set, the first profile data Set and preparation that are difficult to control. It is frequently including a plurality of members, each member being a the case that one batch of a nutraceutical may be effective, quantitative measure of the amount of a distinct RNA or there is no assurance that a Second batch will be effective. protein constituent in a panel of constituents Selected So that Moreover, analysis of nutraceuticals is problematic because measurement of the constituents enables measurement of the these drugs are complex mixtures in which little is known biological condition; and producing a calibrated profile data with respect to the active agent. set for the panel, wherein each member of the calibrated profile data Set is a function of a corresponding member of 0005 All new therapeutic agents require some form of the first profile data Set and a corresponding member of a clinical trials. It is known that a drug for treating tumor that baseline profile data Set for the panel, the calibrated profile is tested in a clinical trial using Standard recruiting tech data Set providing a measure of the biological condition as niques for patients, may in fact show only limited efficacy. affected by the agent. If the beneficial effect observed in a clinical population is too Small, the drug will not receive approval by the Food and 0009. In a another embodiment, a method is provided for Drug Administration for use in the population at large. evaluating the effect on a biological condition by a first agent However, the small beneficial effect observed may in fact be in relation to the effect by a Second agent, including: an artifact of the clinical trial design or the clinical endpoint obtaining, from first and Second target populations of cells to in the population of patients. It would be desirable to have which the first and Second agents have been respectively US 2004/0225449 A1 Nov. 11, 2004 administered, first and Second Samples respectively, each which has been administered an agent, the Sample having at Sample having at least one of RNAS and proteins, deriving least one of RNAS and proteins, each profile data Set from the first sample a first profile data set and from the includes a plurality of members, each member being a Second Sample a Second profile data Set, the profile data Sets quantitative measure of the amount of a distinct RNA or each including a plurality of members, each member being protein constituent in a panel of constituents Selected So that a quantitative measure of the amount of a distinct RNA or measurement of the constituents enables measurement of a protein constituent in a panel of constituents Selected So that biological condition; and the panel is the Same for all profile measurement of the constituents enables measurement of the data Sets. biological condition; and producing for the panel a first 0014. In a another embodiment of the invention, a calibrated profile data Set and a Second profile data Set, method is provided for evaluating a biological condition of wherein (i) each member of the first calibrated profile data a Subject, based on a Sample from the Subject, the Sample Set is a function of a corresponding member of the first having at least one of RNAS and proteins, the method profile data Set and a corresponding member of a first including: deriving from the Sample a first instance of a baseline profile data set for the panel, and (ii) each member profile data Set, the profile data Set including a plurality of of the Second calibrated profile data Set is a function of a members, each member being a quantitative measure of the corresponding member of the Second profile data Set and a amount of a distinct RNA or protein constituent in a panel corresponding member of a Second baseline profile data Set of constituents Selected So that measurement of the constitu for the panel, the calibrated profile data Sets providing a ents enables measurement of the biological condition; and measure of the effect by the first agent on the biological producing a first instance of a calibrated profile data Set for condition in relation to the effect by the Second agent. the panel, wherein each member of an instance of the 0010. In a further embodiment, a method of conducting a calibrated profile data Set is a function of a corresponding clinical trial of an agent, is provided, including: causing the member of an instance of the profile data Set and a corre blind administration of a Selected one of a placebo and the sponding member of an instance of a baseline profile data Set agent to each candidate of a pool of Subjects, and using for the panel, the calibrated profile data Set providing a quantitative gene expression to monitor an effect of Such measure of the biological condition of the Subject; accessing administration. data in a condition database, the condition database having a plurality of records relating to a population of Subjects, 0011. In another embodiment, a digital storage medium is each record corresponding to a distinct instance of the provided on which is stored a computer readable calibrated calibrated profile dataset; and evaluating the first instance of profile data Set, wherein: the calibrated profile data Set the calibrated profile data Set in relation to data in the relates to a Sample having at least one of RNAS and proteins condition database. derived from a target cell population to which an agent has been administered; the calibrated profile data Set includes a 0015. In another embodiment of the invention, a method first plurality of members, each member being a quantitative is provided of displaying quantitative gene expression analy measure of a change in an amount of a distinct RNA or sis data associated with measurement of a biological con protein constituent in a panel of constituents Selected So that dition, the method including: identifying a first profile data measurement of the constituents enables measurement of a Set pertinent to the gene expression analysis data, the first biological condition as affected by administration of the profile data Set including a plurality of members, each agent. member being a quantitative measure of the amount of a distinct RNA or protein constituent in a panel of constituents 0012. In another embodiment, a digital storage medium is Selected So that measurement of the constituents enables provided on which is Stored a plurality of records Ri relating measurement of the biological condition; producing a cali to a population of Subjects, each record R corresponding to brated profile data Set for the panel, wherein each member a distinct instance P of a computer readable profile data set of the calibrated profile data Set is a function of a corre P wherein: each instance P of the profile data set P relates sponding member of the first profile data Set and a corre to a distinct Sample derived from a Subject, the Sample sponding member of a baseline profile data Set for the panel, having at least one of RNAS and proteins; the profile data P the calibrated profile data Set providing a measure of the set includes a plurality of members M, each member M, biological condition of the Subject; and displaying the cali being a quantitative measure of the amount of a distinct brated profile data Set in a graphical format. RNA or protein constituent in a panel of constituents Selected So that measurement of the constituents enables 0016. Another embodiment is directed to a descriptive measurement of a biological condition; each record R. record of a change in a biological condition, that includes: includes, for each member M of a corresponding distinct a first Set of numerical gene expression values for a panel of instance P of the profile data Set P, a value corresponding to gene loci, each value in the Set corresponding to a single the value of the member Mii; and each record R.; also gene locus in a panel of gene loci, the Set of values forming includes a reference to a characteristic of the Subject relative a profile data Set for a population of cells Subjected to a first to the record, the characteristic being at least one of age biological condition; a Second set of numerical gene expres group, gender, ethnicity, geographic location, diet, medical Sion values for the panel of gene loci, each value in the Set disorder, clinical indicator, medication, physical activity, corresponding to a Single gene locus, the Set of values body mass, and environmental exposure. forming a baseline profile data Set for a Second population of cells Subjected to a Second biological condition, the Second 0013 In a further embodiment, a digital storage medium Set of values optionally being an average for multiple gene is provided on which is Stored a large number of computer expression values from multiple populations of cells for readable profile data Sets, wherein each profile data Set each locus in the panel; and a third Set of numbers corre relates to a Sample derived from a target cell population to sponding to the ratio of the first Set of values and the Second US 2004/0225449 A1 Nov. 11, 2004
Set of values with respect to each gene locus in the panel, the 0023. In another embodiment, a method is provided for third Set being a calibrated profile data Set, the profile data accessing biological information on a digital Storage Set and the calibrated profile data Set being descriptive of the medium as described above, including: making the infor first biological condition with respect to the Second biologi mation available to a user. cal condition. 0024. In another embodiment, a method is provided for 0.017. In another embodiment, a method for diagnosing a consumer evaluation of a product, wherein the consumer biological condition of a Subject is provided that includes: evaluation is dependent on a signature profile, including: obtaining a Sample from a Subject, Subjecting a population identifying the product using the Signature profile. of cells to the Sample and determining the presence of a first 0025. In another embodiment, a computer program prod biological condition with respect to a Second biological uct is provided for evaluating a biological condition of a condition according to any of the above claims. Subject or for evaluating a biological condition resulting from the use of an agent, including a computer usable 0.018. In another embodiment, a method is provided for medium having computer readable program code thereon, diagnosing a Susceptibility for a biological condition in a the computer program code, including: a program code for Subject, that includes obtaining a Sample from the Subject; classifying a Sample from the Subject or the agent for an creating a descriptive record, according to the above, identifiable record; a program code for deriving a first data wherein the baseline Set of values is an average of Second Set, the first profile data Set including a plurality of members, values contained in a library of descriptive records for the each member being a quantitative measure of the amount of Second biological condition; the library containing a plural a distinct RNA or protein constituent in a panel of constitu ity of descriptive records grouped according to a predeter ents Selected So that measurement of the constituents enables mined biological condition; comparing the calibrated profile measurement of the biological condition; the profile data Set data set of the subject with the library of calibrated profile being Stored in the record; and a program code for optionally data Sets and diagnosing the Susceptibility of the Subject. producing a calibrated profile data Set for the panel, for 0019. In another embodiment, a method is provided for Storage in the record, each member of the calibrated profile monitoring the progreSS of a biological condition, including: data Set being a function of a corresponding member of the creating a plurality of descriptive records, according to the first profile data Set and a corresponding member of a above, wherein each Set of first values is determined at baseline profile data Set for the panel, the calibrated profile preselected time intervals with respect to the first record; data Set providing a measure of the biological condition of comparing each calibrated profile data Set with a library of the Subject. calibrated profile data Sets, the plurality of calibrated profile 0026. In another embodiment of the invention, a com data Sets being grouped according to a predetermined bio puter System for evaluating a biological condition of a logical condition; and determining the progreSS of the bio Subject or for evaluating a biological condition resulting logical condition with respect to gene expression. from the use of an agent is provided, the computer System, including: a classification module for classifying a Sample 0020. In another embodiment, a method is provided for from the Subject or the agent in an identifiable record; a establishing the biological activity of a composition, includ derivative module for deriving a first data set, the first profile ing: Selecting a population of cells; Subjecting the cells to the data Set including a plurality of members, each member composition; and determining the record according to the being a quantitative measure of the amount of a distinct above description using a Standardized baseline profile data RNA or protein constituent in a panel of constituents Set for the biological condition. Selected So that measurement of the constituents enables 0021. In another embodiment, a method is provided for measurement of the biological condition; and a production determining which therapeutic agent from a choice of a module for producing a calibrated profile data Set for the plurality of therapeutic agents to administer to a Subject So panel, wherein each member of the calibrated profile data Set as to change a biological condition in a Subject from a first is a function of a corresponding member of the first profile biological condition to a Second biological condition; data Set and a corresponding member of a baseline profile including: Subjecting a Sample from the Subject to each of a data Set for the panel, the calibrated profile data Set provid plurality of therapeutic agents, determining a descriptive ing a measure of the biological condition of the Subject. record for each of the Samples according to any of the above 0027. In another embodiment, a method is provided for described methods, comparing each of the calibrated profile analyzing a patient for a biological condition at a remote data Sets to a library of calibrated profile data sets, the library Site, including: providing a kit for measuring a profile data of calibrated data Sets being grouped according to a prede base for evaluating a biological condition, the kit including termined biological condition; and determining which of the reagents for quantitative analysis of RNA or protein for a therapeutic agents is capable of changing the first biological panel of gene loci, accessing a centralized database contain condition in the Subject to the Second biological condition in ing baseline profile data Sets corresponding to the panel; the Subject. determining the calibrated profile data Set for the patient; 0022. In another embodiment, a method is provided for and analyzing the biological condition of the patient. characterizing the biological effectiveness of a Single batch 0028. Further embodiments of the invention include the of a composition produced by a manufacturing process, use of calibrated profile data bases for determining the comprising: providing a fingerprint or Signature profile biological condition at one site in a Subject from a Sample according to any of the above methods, and labeling the taken from a Second remote site. The biological condition batch of the composition by placing the fingerprint (signa may include disease, therapeutic interventions, aging, health ture profile) on each container in the batch. conditioning and exercise, exposure to toxins, Status of US 2004/0225449 A1 Nov. 11, 2004
infection and health Status. For example, calibrated precision ing member of a baseline profile data Set for the profiles may be used to measure a biological condition(s) in panel, the calibrated profile data Set providing a one site (for example, the liver) by Sampling cells from the measure of the biological condition of the Subject. Same Subject, but at a different site not generally considered a target for the biological condition, for example, peripheral 0039. In this embodiment, the biological condition relates blood cells in the case of liver disease. to inflammation and the panel includes at least half, and, optionally, at least eighty percent of the constituents of the 0029 Further embodiments of the invention include the Inflammation Selected Panel of Table 1. In a related embodi use of calibrated profile data bases for determining the ment, the biological condition relates to cell growth and biological condition of the Subject that includes placing a differentiation and the panel includes at least half, and cell or fluid Sample on indicator cells to assess the biological optionally at least eighty percent, of the constituents of the condition, the biological condition including disease, thera Cell Growth and Differentiation Selected Panel of Table 2. peutic interventions, aging, health conditioning and exer In other related embodiments, the biological condition cise, exposure to toxins, Status of infection and health Status. relates to metabolism and toxicity and the panel includes at 0030) Further embodiments of the invention include the least half, and optionally at least eighty percent, of the use of calibrated profile data bases and profiles to assess, constituents of the Liver Metabolism and Toxicity Selected compare and contrast the bioactivities of therapeutic agents Panel of Tables 3 or 7. In another related embodiment, the and therapeutic agent candidates including comparison of biological condition relates to skin response and the panel two agents having unknown properties, comparison of includes at least half, and optionally at least eighty percent, agents that are complex mixtures against those that are of the constituents of the Skin Response Selected Panel of Simple mixtures and comparisons of a single agent against a Table 4. In another related embodiment, the biological class of agents. condition relates to the vascular System and the panel includes at least half, and optionally, at least eighty percent, 0.031 Further embodiments of the invention include the of the constituents of the Vascular Selected Panel of Table 6. use of calibrated profile databases derived from in vitro In a further related embodiment, the biological condition dosing of an agent in indicator cells, or fluids or cells ex vivo relates to the prostate health and disease and the panel to predict in Vivo activities, activities including efficacy and includes at least half, and optionally at least eighty percent toxicity and further permitting data on Short term in vivo of the constituents of the Prostate Selected Panel of Table 5. dosing of agent to predict long-term activities as described herein. 0040 Another embodiment of the invention provides a method, for evaluating a biological condition of a Subject, 0032) Another embodiment of the invention is at least that includes: obtaining from the Subject a Sample having at one databases and its uses, the databases containing at least least one of RNAS and proteins, deriving from the Sample a one of calibrated profile data Sets and baseline profile data profile data Set, the profile data Set including a plurality of Sets for discrete populations identified according to factors members, each member being a quantitative measure of the including diseases, geography, ethnicity, age and State of amount of a distinct RNA or protein constituent in a panel health. of constituents Selected So that measurement of the constitu 0033. A further embodiment of the invention is a data ents enables measurement of the biological condition; base corresponding to an individual over time, the uses wherein Such measurement is performed for each constituent including managing a personalized health care program. under conditions wherein efficiencies of amplification for all constituents are Substantially similar, the profile data Set 0034. Additional embodiments include methods of run providing a measure of the biological condition of the ning a clinical trial using calibrated profile data and data Subject. bases containing calibrated profile data from in vitro and in Vivo Studies of the effect of the agent on populations of cells 0041 Another embodiment of the invention provides a and methods of building a clinical research network that method, for evaluating a biological condition of a Subject, uses calibrated profile data and traditional medical data. that includes: obtaining from the Subject a first Sample having at least one of fluid, cells and active agents, applying 0.035 Another embodiment of the invention provides a the first Sample or a portion thereof to a defined population method, for evaluating a biological condition of a Subject. of indicator cells, obtaining from the indicator cells a Second This method includes: Sample containing at least one of RNAS or proteins, deriving 0036 a. obtaining from the subject a sample having from the Second Sample a profile data Set, the profile data Set at least one of RNAS and proteins; including a plurality of members, each member being a quantitative measure of the amount of a distinct RNA or 0037 b. deriving from the sample a first profile data protein constituent in a panel of constituents Selected So that Set, the first profile dataSet including a plurality of measurement of the constituents enables measurement of the members, each member being a quantitative measure biological condition, wherein Such measure is performed for of the amount of a distinct RNA or protein constitu each constituent under conditions wherein efficiencies of ent in a panel of constituents Selected So that mea amplification for all constituents are Substantially similar, Surement of the constituents enables measurement of the profile data Set providing a measure of the biological the biological condition; and condition of the Subject. 0038 c. producing a calibrated profile data set for 0042 Another embodiment of the invention provides the panel, wherein each member of the calibrated method for evaluating a biological condition affected by an profile data Set is a function of a corresponding agent, the method that includes obtaining, from a target member of the first profile data Set and a correspond population of cells to which the agent has been administered, US 2004/0225449 A1 Nov. 11, 2004 a Sample having at least one of RNAS and proteins, deriving 0051 FIG. 6 is a diagram showing the creation of a from the Sample a profile data Set, the profile data Set library of profile data associated with a plurality of Subjects including a plurality of members, each member being a in accordance with an embodiment of the present invention. quantitative measure of the amount of a distinct RNA or protein constituent in a panel of constituents Selected So that 0052 FIG. 7 is a diagram illustrating the structure of a measurement of the constituents enables measurement of the profile data record in accordance with an embodiment of the biological condition; wherein Such measure is performed for present invention. each constituent under conditions wherein efficiencies of 0053 FIG. 8 is a diagram illustrating a data entry screen amplification for all constituents are Substantially similar, for a data record of the type shown in FIG. 7 and typical the profile data Set providing a measure of the biological contexts in which data records may be compiled in accor condition as affected by the agent. dance with embodiments of the present invention. 0043. Efficiencies of amplification of all constituents may 0054 FIG. 9 shows an embodiment of the present inven differ by less than approximately 2%. The efficiencies of tion in which profile data, in either the raw or calibrated amplification may differ by less than approximately 1%. form, is evaluated using data from a database that is Moreover, in any of the embodiments of the invention remotely accessed over a network. described above which refers to a panel, the panel may include at least four constituents Selected from any one of 0055 FIG. 10 shows a schematic of a phase two clinical Tables 1 through 7. For example, at least four constituents trial that utilizes gene expression profiling (a). The right may be selected from the group consisting of expression hand panel (b) indicates that the same information may be products of TNF-C., IL-1-C, IL-8, IFN-y, IL-8, and IL-10. used in Phase IV or post marketing Studies to compare the efficacy of already approved and marketed drugs or to guide 0044) In another embodiment of the invention, a kit is the marketing of Such therapies, to guide the choice of provided having primer-probe combinations for measuring therapy for an individual Subject or population from within expression products of at least four constituents Selected a class of appropriate compounds. from any one of Tables 1 through 7. The kit may further include a primer probe combination constructed So as to 0056 FIG. 11 is a bar graph that shows a graphical hybridize only to at least one of cDNA and mRNA at a representation in the form of a histogram representing biologically relevant locus. Moreover, in each combination, calibrated profile data Sets based on quantitative expression a reverse primer may be selected which is complementary to of RNA in cells of a whole blood sample using a panel of 12 a coding DNA Strand located across an intron-exonjunction, constituents where each constituent corresponds to a unique with not more than three bases of a three-prime end of the gene locus. (a) The blood sample is stimulated ex vivo with reverse primer being complementary to a proximal eXon. heat killed Staphylococci are further exposed H7-TPCK, H9-UT-77, or H16-Dex as indicated. The baseline profile BRIEF DESCRIPTION OF THE DRAWINGS data set is a blood sample stimulated ex vivo (in vitro) with 004.5 The foregoing features of the invention will be heat killed Staphylococci (b) The blood sample is stimulated more readily understood by reference to the following ex vivo with lipopolysaccharide (LPS) and is then further detailed description, taken with reference to the accompa exposed to compounds H7-TPCK, H9-UT-77, or H16-Dex nying drawings, in which: as indicated. 0.046 FIG. 1 is a diagram showing the flow of informa 0057 FIG. 12 is a bar graph with a logarithmic axis that tion from data acquired in molecular pharmacology and shows a graphical representation of calibrated profile data toxicology, clinical testing, and use of the data for the sets for whole blood stimulated ex vivo with lipopolysac application to individualized medicine. charide (LPS), using a panel of 9 constituents, each con Stituent corresponding to a gene locus encoding the gene 0047 FIG. 2 is a diagram showing the drug discovery products indicated, the blood being further exposed to pathway of new compounds from early leads to likely drug anti-inflammatory agents: methotrexate, meclofenamate and candidates. Although calibrated profile data Sets are indi methylprednisolone. The baseline profile data set is derived cated at the pre-clinical Step, gene expression data can be acquired and is useful at any of the Stages shown. IND refers from LPS stimulated (but otherwise untreated) cells. to investigative new drug and refers to an early Stage in 0058 FIG. 13 are bar graphs with a logarithmic axis that regulatory review. shows a graphical representation of calibrated profile data sets for two different samples of whole blood (a)991116 and 0.048 FIG. 3 is a diagram presenting a comparison of in (b) 991028 reflecting the biological condition of the cells vivo and in vitro protocols for forming calibrated profile using a panel of 24 members, each member corresponding data Sets for rapidly assessing product candidate toxicity and to a gene locus, the baseline profile data Set being derived efficacy in accordance with Several embodiments of the from untreated cells. The calibrated data sets for cells present invention. exposed for Six hours to three inflammation inducing agents 0049 FIG. 4 is a diagram showing the application of (lipopolysaccharide, heat killed Staphylococci, and phyto gene expression profiling as a guide to pre-clinical and hemagglutinin) are compared for each Sample. (c) shows a clinical Studies in accordance with an embodiment of the direct comparison of LPS stimulated 991116 with respect to present invention. 991028 as the baseline profile data set (d) shows a direct 0050 FIG. 5 is a diagram showing a method in accor comparison between unstipulated 991116 and 991028. dance with an embodiment of the present invention for 0059 FIG. 14 is a bar graph with a logarithmic axis that obtaining profile data in the absence of a Stimulus and in the shows a graphical representation of calibrated profile data presence of a Stimulus. Sets using a panel of 22 constituents, each constituent US 2004/0225449 A1 Nov. 11, 2004 corresponding to a gene locus, the baseline profile data Set baseline for a non-Smoker. (b) shows a calibrated profile data being derived from untreated cells. Whole blood is exposed Set for a Subject with chronic obstructive pulmonary disease for six hours ex vivo to three inflammation inducing agents against a baseline for a Subject lacking this disease. The (lipopolysaccharide, heat killed Staphylococci, and phyto baseline profile data Set is derived from a Subject that is hemagglutinin) which are then treated with a single anti “normal” with respect to these conditions. inflammatory agent (methyl prednisolone) to reveal Simi larities and differences in the effect of a single agent on cell 0065 FIG. 20 illustrates that an individual responses can populations differing in their biological condition. be distinguished from a similarly treated population. A comparison of the response of a single animal compared to 0060 FIG. 15 is a bar graph with a logarithmic axis that its experimental cohort (n=5 animals) with respect to a shows a graphical representation of calibrated profile data single locus (GST-P) is provided. The baseline data set is the sets for whole blood where one calibrated data set refers to cohort average. The figures shows that this animal varied a subject (subject 2) who has been treated in vivo with a Significantly from the daily, population average in the first corticosteroid (dexamethasone), a second data set refers to two days of the study, but became more similar to the cohort the treatment of a blood Sample from the same Subject prior average with time after treatment with acetaminophen. to in Vivo treatment where that Sample has been treated eX vivo (in vitro) and the third data set refers to a second subject 0066 FIG. 21 is a bar graph with a logarithmic axis that treated in vivo with dexamethasone (subject 1). The data sets shows a graphical representation of calibrated profile data demonstrate the reproducibility and predictability of an eX sets for samples of blood treated ex vivo with LPS or LPS vivo (in vitro) treatment of blood compared to in vivo and one of three anti-inflammatory herbals (Echinacea, treatment with the same agent. The figure also shows minor Arnica or Siberian Ginseng) at a concentration of 200ug/ml. variation between Samples from different Subjects reflecting A panel of 24 constituents is used. The baseline profile data interpersonal variability. A panel of 14 constituents is pro set is derived from LPS stimulated cells absent a herbal vided. The baseline profile data set is derived from untreated treatment. The figure illustrates the effectiveness of the use whole blood from the cognate Subject. of the calibrated selected profile to investigate the overall effects of complex compounds Such as nutraceuticals whose 0061 FIG. 16 is a bar graph with a logarithmic y axis that biological effect is a Summation of more than one activity. In shows a graphical representation of calibrated profile data this case, each of the herbals is consumed as an immuno sets for whole blood where one calibrated data set refers to Stimulant, however the calibrated Selected profiles reveal a (a) 2 subjects who have been treated in vivo with an inactive unique pattern shows a mixture of both immunostimulatory placebo for 3 days and (b) active prednisolone for 3 days at and anti-inflammatory effects. 100 mg/day. The data set shows some variation between Samples from different Subjects treated with the same drug. 0067 FIG. 22 is a bar graph with a logarithmic axis that The data Sets demonstrate Similarity of responses across the shows a graphical representation of calibrated profile data Same gene loci, as well as, quantitative variation at other loci sets for samples of blood treated ex vivo with LPS or LPS Suggesting quantifiable interpersonal variation. A panel of and methylprednisolone or LPS and Arnica. The baseline eight members is provided. The baseline profile data Set is profile data set is LPS treated blood sample. derived from untreated whole blood. 0068 FIG. 23 is a bar graph with a logarithmic axis that 0.062 FIG. 17 is a bar graph with logarithmic y axis that shows a graphical representation of calibrated profile data shows a graphical representation of calibrated Selected pro sets for samples of THP-1 cells treated with LPS or LPS and file data Sets for two Samples taken from a single Subject Arnica at three different concentrations using a panel of 22 within a 19 day period using a panel (e.g., inflammation constituents. The baseline profile data set is untreated THP-1 panel) of 24 members where each member corresponds to a cells. The figure illustrates a concentration response with unique gene locus. The baseline profile data Set relates to respect to the gene expression acroSS the calibrated profile. peripheral blood taken from the Subject prior to treatment. 0069 FIG. 24 is a bar graph with a logarithmic axis that 0063 FIGS. 18(a) through 18(e) are bar graphs with a shows a graphical representation of calibrated profile data logarithmic axis that Show a graphical representation of sets for samples of THP-1 cells treated ex vivo with four calibrated profile data sets for each of 5 subjects from which different commercial brands of Echinacea using a panel of 8 a blood Sample has been taken. Each of the blood Samples constituents. The baseline profile data set is untreated THP-1 was exposed to the inflammatory agent phytohemagglutinin cells. (PHA) or to a therapeutic agent (anti-inflammatory agent) at different concentrations: 0.1 uM, 0.3 uM, 1 uM, 3 uM and 0070 FIG. 25 illustrates the use of the calibrated profile 5uM, for a 4 hour period ex vivo (in vitro) so as to determine to compare relative efficacy acroSS brands, or different the optimum dose for treating the Subject. A panel of 6 formulations. Calibrated profile data sets for herbal prepa constituents was used corresponding to 6 gene loci. The rations from different manufacturing Sources with respect to baseline profile data Set was an untreated Sample obtained an indicator monocytic cell line (THP-1) are shown graphi from the cognate donor. cally, the baseline profile data set being THP-1 cells absent the herbal. (a) Three commercial herbal Echinacea prepa 0.064 FIG. 19 is a bar graph with a logarithmic axis that rations at 250 (ug/ml); (b) three herbal preparations at shows a graphical representation of calibrated profile data different concentrations (250 tug/ml, 50 tug/ml and 3-10 sets for three different subjects having different biological Aug/ml) (c) four commercial Echinacea brands at 250 ug/ml). conditions using a panel with 24 constituents. The profile data Sets show variability according to these conditions 0071 FIGS. 26(a) through 26(d) illustrate calibrated pro providing the basis for a diagnostic Signature panel: (a) file data sets, using a Subset of the Inflammation Selected shows a calibrated profile data Set for a Smoker against a Panel, that show the effect of administration of a steroid. US 2004/0225449 A1 Nov. 11, 2004
0072 FIGS. 27(a) through 27(d) illustrate calibrated pro cell cultures of eukaryotic or prokaryotic cells. Cell lines can file data sets, using a Subset of the Inflammation Selected be primary cultures or cell Samples, e.g. from a tumor, from Panel, providing a comparison of the effects of administra blood or a blood fraction, or biopsy explants from an organ, tion of methylprednisolone and Ibuprofen. or can be established cell lines or microbial Strains. 0073 FIGS. 28(a) through 28(d) illustrate calibrated pro 0082. A “region of the subject” from which proteins are file data sets, using a Subset of the Inflammation Selected obtained may (but is not required to be) the same part of the Panel, in identifying chronic obstructive pulmonary disease subject from which has been obtained a collection of cells or (COPD) patients. a population of cells. The cells and the proteins may both be obtained from blood of the subject, for example. Alterna 0074 FIGS. 29(a) and 29(b) provide illustrations in tively, for example, the cells may be obtained from blood which evaluations of the effects of drug exposure performed and the proteins may be obtained from a Scraping of tissue in Vitro correspond closely with evaluations performed in or Vice versa. Similarly, the proteins may be obtained from Vivo, employing in each case calibrated profile data sets, urine of the Subject, for example, whereas the cells may be using a subset of the Inflammation Selected Panel. obtained elsewhere, as, for example, from blood. 0075 FIG. 30 illustrates the effect of different agents evaluated using a subset of the Selected Prostate Panel, and 0083. A “panel” of genes is a set of genes including at shows broad functions of constituents of the panel. least two constituents. 0076 FIG.31 illustrates the effect of the pharmaceutical 0084. A “normative” condition of a subject to whom a agent, clofibrate, as measured on a rat liver metabolism composition is to be administered means the condition of a Selected panel. The profiles for Six rats are provided as Subject before administration, even if the Subject happens to indicated on the Z axis. The control (baseline) is a set of rats be Suffering from a disease. treated only with the carrier control. 0085. An “expression” of a gene includes the gene prod 0077 FIG.32 illustrates the ability of the rat metabolism uct whether RNA or protein resulting from translation of the Selected panel to differentiate drug responses (clofibrate messenger RNA. versus benzoapyrene) in Spracque-Dawley rats. Clofibrate 0086 A“large number of data sets based on a common (right hand bars) and Benzopyrene (left hand bars). The panel of genes is a number of data Sets Sufficiently large to control (baseline) is a set of rats treated only with the carrier permit a Statistically significant conclusion to be drawn with control. FIG. 33 illustrates the effect of administration of a respect to an instance of a data set based on the same panel. stimulant (TNF-C) as measured by a combination of con Stituents Selected from the inflammation, Skin/epithelial, and 0087. A “biological condition” of a subject is the condi vascular Selected panels. The target is human keratinocytes tion of the Subject in a pertinent realm that is under obser in culture. The baseline is non stimulated cells. The baseline Vation, and Such realm may include any aspect of the Subject is a set of rats that were non-stimulated. FIG. 34 illustrates capable of being monitored for change in condition, Such as the effect of administration of benzoapyrene on cryo health, disease including cancer, trauma, aging; infection; preserved human hepatocytes over time as measured by the tissue degeneration; developmental Steps, physical fitness, human liver Selected panel. The control (baseline) are cells obesity, or mood. AS can be seen, the conditions may be treated similarly but without the addition of benzoapyrene. chronic or acute or simply transient. Moreover, a targeted FIG. 35 demonstrates the effect of treating human umbilical biological condition may be manifest throughout the organ vein endothelial cells in culture with TNFC. for 24 hours. ism or population of cells or may be restricted to a specific The control or baseline is established from cells handled organ (Such as Skin, heart, eye or blood). The term “bio similarly but without the addition of the stimulant. logical condition' includes a "physiological condition'. 0078 FIG. 36 illustrates the protective effect of the 0088. The “blind administration” of a selected one of a antioxidant n-acetylcysteine (NAC) on human keratinocytes composition or placebo to a Subject in a clinical trial in culture after exposure the UVB energy. The dark bars involves administering the composition or placebo to the indicate the effect of UVB exposure only. Cells that were Subject in accordance with a protocol pursuant to which the treated with NAC followed by exposure to the same UVB Subject lacks knowledge whether the Substance administered energy show a decreased induction of expression at most of is the composition or a placebo. the gene loci covered by the skin Selected panel. The 0089 An “organism' is any living cell including micro baseline corresponds to cells exposed to assay media only. organisms, animals and plants. An animal is commonly in this context a mammal, but may be a vertebrate non DETAILED DESCRIPTION OF SPECIFIC mammal, as e.g., a Zebra fish, or an invertebrate, as, e.g. EMBODIMENTS Caenorhabditis elegans. 0079 AS used in this description and the accompanying 0090 An “agent” is a composition or a stimulus. A claims, the following terms shall have the meanings indi “stimulus' may include, for example ultraviolet A or B, or cated, unless the context otherwise requires: light therapy for Seasonal affective disorder, or treatment of 0080 A“collection of cells” is a set of cells, wherein the pSoriasis with psoralen or treatment of melanoma with Set has at least one constituent. embedded radioactive Seeds, other radiation exposure, etc. 0081. A “population of cells” includes one or more cells. 0091 A“composition” includes a chemical compound, a A population of cells may refer to cells in Vivo or to in vitro nutraceutical, a pharmaceutical, a homeopathic formulation, cultures. In Vitro cultures may include organ cultures or cell an allopathic formulation, a naturopathic formulation, a cultures where cell cultures may be primary or continuous combination of compounds, a toxin, a food, a food Supple US 2004/0225449 A1 Nov. 11, 2004
ment, a mineral, and a complex mixture of Substances, in but rather a correlation may be established by evaluating a any physical State or in a combination of physical States. panel of constituents of reasonable size (for example up to 100 constituents) and iteratively testing the gene expression 0092. A “clinical indicator” is any physiological datum profiles for different subjects or for the same subject from used alone or in conjunction with other data in evaluating the which the most informative loci for a particular condition physiological condition of a collection of cells or of an may be selected. An informative Subgroup of constituents in organism. This term includes pre-clinical indicators. a panel may be Selected that consistently vary for a particular 0093. A “selected panel” is an experimentally verified set condition and this Subgroup may then become the Signature of constituents, each constituent being a distinct expressed panel, the Signature panel giving rise to a signature profile. product of a gene, whether RNA or protein, wherein con Stituents of the Set are Selected So that their measurement 0100. In another embodiment of the invention, the profile provides a measurement of a targeted biological condition A data Sets by themselves describe a biological condition or an "Selected profile' is a Set of values associated with constitu effect of an agent on a biological condition. A profile data Set ents of a Selected panel resulting from evaluation of a represents a set of gene expression values that correspond to biological Sample (or population of Samples). particular gene constituents in a panel of constituents, wherein the panel may be two or more constituents Selected 0094. A “signature profile” is an experimentally verified to include gene loci that directly or indirectly vary with a Subset of a Selected profile Selected to discriminate a bio particular biological condition, and wherein the gene expres logical condition, agent or physiological mechanism of Sion values themselves are informative. This approach does action. A “Signature panel” is a Subset of a Selected panel, the not require comprehensive analysis of all gene expression in constituents of which are Selected to permit discrimination target cells associated with a particular condition. Nor is any of a biological condition, agent or physiological mechanism one Single gene locus necessarily of particular Significance. of action. Rather a pattern of expression (a profile) is Sought that correlates, in a reproducible manner, with a particular con 0.095 “Distinct RNA or protein constituent” in a panel of dition. There may be no a priori knowledge of a correlation constituents is a distinct expressed product of a gene, but rather a correlation may be established by evaluating a whether RNA or protein. panel of constituents of reasonable size (for example up to 0.096 “Precision” as used herein, means the repeatability 100 constituents) and iteratively testing the gene expression and reproducibility of an assay for gene expression in terms profiles for different subjects or for the same subject from of variance or standard deviation where repeatability is the which the most informative loci for a particular condition measure of variance of a Single Sample measured many may be Selected. AS described above, an informative Sub times and reproducibility is the variance accounting for group of constituents in a panel may be selected that assaying Similar biological conditions (i.e., Similar Samples) consistently vary for a particular condition and this Subgroup using multiple instruments, reagent lots, and multiple opera may then become the Signature panel, the Signature panel tors over time. Repeatability is Sometimes referred to as giving rise to a signature profile. intra-assay variation and reproducibility may be referred to 0101. In further embodiments of the invention, any cali as inter-assay variability. Reproducibility describes the vari brated data set for an individual that has more members than ance around Similar Samples obtained from a population of reflective of a single signature panel may be mined for individuals with a similar biological condition including calibrated profiles that correspond to additional Signature normal, good health or clinically definable disease States. panels, thereby potentially providing new insights into 0097 “Substantially reproducible” as used herein means mechanisms of action of a biological condition on Sets of that the variance, as measured by coefficient of variation or genes. Measurement of changes in transcribed RNA in a cell Standard deviation, for repeatability and reproducibility is as a result of an environmental change or aging is an less than about 3%. exquisitely Sensitive measure of the response of a cell. Techniques available today to quantify transcribed RNA in 0.098 “Reproducible” as used herein means that each a cell add to the sensitivity of the approach. Embodiments of member of a profile data Set is reproducible within a range the invention that are directed to patterns of change in for repeatability and reproducibility of within a range of amounts of transcribed RNA provide a means to focus and 20%, and more particularly within a range of 10%. interpret this rich information. 0099. An embodiment of the invention includes the for 0102) In contrast to the above approach, much attention mation of profile data Sets and calibrated data Sets that in the prior art has been directed to the Sequencing of the describe a biological condition or an effect of an agent on a human genome and the identification of all the genes biological condition. A profile data Set represents a Set of encoded therein. Accompanying the growing amount of values that correspond to measurements of gene expression Sequence data, microarrays provide a means to Survey many wherein the measurement values themselves are informa hundreds to thousands of gene Sequences. Microarrays are tive. A calibrated data Set represents a set of values that being used to provide DNA profiles that identify mutations correspond to variations in gene expression where the varia in an individual and those mutations will be associated with tions are informative. This approach does not require com predictions concerning development of disease in those prehensive analysis of all gene expression in target cells individuals. asSociated with a particular condition. Nor is any one single gene locus necessarily of particular significance. Rather a 0103) Transcriptomics and proteomics are now the focus pattern of expression or variation (a profile) is sought that of increasing attention. These Studies are directed to ana correlates, in a reproducible manner, with a particular con lyzing the entire body of RNA and protein produced by dition. There may be no a priori knowledge of a correlation living cells. Microarrays provide a method for analyzing US 2004/0225449 A1 Nov. 11, 2004
many thousands of different human RNAS as to whether they precision-is informative of the biological condition of the are expressed and by which cells. For example, a project Subject with respect to which gene expression has been undertaken by the National Cancer Institute and others to measured. Further discussion of these points also occurs in examine mRNAS produced by various types of cancer cells, our co-pending application, U.S. application Ser. No. have revealed 50,000 genes that are active in one or more 10/742,458, filed Dec. 19, 2003, which is hereby incorpo cancers. The goal of these Studies is to identify novel cancer rated by reference. That application, points out, for example, drugs that are directed to knocking out or enhancing the that algorithms can be fashioned that use Selected profile production of certain proteins. (Kathryn Brown, The Human data to provide Single-valued indices that are informative, Genome Business Today, Scientific American, July 2000, for example, of health or disease of a Subject. But as shown p.50; Julia Karow, The “Other Genomes, Scientific Ameri can, July 2000, p.53; Ken Howard, “The Bioinformatics there and herein, these matters can also be seen directly in Gold Rush, Scientific American, July 2000, p.58; Carol the Selected profile data. Ezzell, Beyond the Human Genome, Scientific American, 0106 We have exemplified the use of selected panels of July 2000, p.64; all incorporated by reference.) Major efforts constituents corresponding to gene loci from which quanti in correlating genetic variation of individuals and the func tative gene expression is determined by, for example, quan tional interrelationships of genes in health and disease are titatively measuring the transcribed RNA in a Sample of a being conducted in a variety of consortia including the Subject, for applications that include: (a) measurement of Single nucleotide polymorphism consortium and the Human therapeutic efficacy of natural or Synthetic compositions or Epigenome Consortium (Beck et al. Nature BioTechnology stimuli that may be formulated individually or in combina 17 (1999) p 1144). The Epigenome Consortium plans to tions or mixtures for a range of targeted physiological analyze Sets of genome fragments from both healthy and conditions; (b) predictions of toxicological effects and dose diseased individuals in the 500 different human tissues effectiveness of a composition or mixture of compositions (Bioworld International: Dec. 22, 1999). This approach for an individual or in a population; (c) determining how two Seeks to correlate absolute expression of genes associated or more different agents administered in a Single treatment with a particular condition with the presence of that condi might interact So as to detect any of Synergistic, additive, tion. Examples of prior art that seek to measure gene negative, neutral or toxic activity (d) performing pre-clinical expression in absolute amounts including by Subtractive and clinical trials by providing new criteria for pre-Selecting methods or by determining amounts with respect to house Subjects according to informative profile data Sets for reveal keeping genes or by targeting a single gene expression ing disease Status and conducting preliminary dosage Studies system are U.S. Pat. No. 5,643,765; U.S. Pat. No. 5,811,231; for these patients prior to conducting phase 1 or 2 trials. U.S. Pat. No. 5,846,720; U.S. Pat. No. 5,866,330; U.S. Pat. Gene expression profiling may be used to reduce the cost of No. 5,968,784; U.S. Pat. No. 5,994,076; WO 97/41261; WO phase 3 clinical trials and may be used beyond phase 3 trials, 98/24935; WO 99/11822; WO 99/44063; WO 99/46403; (e) labeling for approved drugs, (f) Selection of Suitable WO 99/57130; WO00/22172 and WO00/11208. medication in a class of medications for a particular patient 0104. We have taken a different and novel approach to the that is directed to their unique physiology, (g) diagnosing or above by identifying reproducible patterns of gene expres determining a prognosis of a medical condition or an infec Sion that are informative by Virtue of the degree of variation tion which may precede onset of Symptoms or alternatively between a Sample and a baseline, for example, in a Subject diagnosing adverse Side effects associated with administra with the condition and a subject without the condition. The tion of a therapeutic agent, (h) managing the health care of variations may be correlated with other non-genetic indica a patient; and (i) quality control for different batches of an tions Such as clinical indicators (for humans) of a traditional agent or a mixture of agents. nature but are not required per Se to be causative. Accord ingly, the amount of gene expression product (for example 01.07 The Subject RNA transcript) produced by a gene locus in a cell under certain circumstances is measured and then Stored as a value 0108. The methods herein can be applied to a subject that in a first profile data set. This value is calibrated with respect includes any living organism where a living organism to a Second value (a baseline profile data set) to provide a includes a prokaryote Such as a bacterium or a eukaryote member of a calibrated profile data set. The values recorded including Single celled eukaryotic organisms at one end of for the profile data Set, relying on a particular baseline data the Spectrum and humans at the other and everything in Set to produce a calibrated data Set, become part of the between including plants. The figures relate to calibrated descriptive record; any or all of these results can be Stored profile data Sets obtained from humans and mammals. in a database which may be accessed through a global Nonetheless, the methods disclosed here may be applied to network. In this way any new data in the form of a profile cells of other organism without the need for undue experi data Set or a calibrated profile data Set measured at any mentation by one of ordinary skill in the art because all cells global location can be directly compared to an archive of transcribe RNA and it is known in the art how to extract descriptive records including calibrated profile data Sets and RNA from all types of cells. baseline data Sets So as to extend the Stored library of profiles 0109) A tissue sample may include a single cell or and provide predictive, diagnostic, or evaluative data about multiple cells or fragments of cells. Body fluid includes a particular biological condition or agent. blood, urine, Spinal fluid, lymph, mucosal Secretions, 0105. As we have exemplified below, selected profiles hemolymph or any other body fluid known in the art for a are in fact of themselves informative of and descriptive of Subject. For an animal Subject, a tissue or fluid Sample may biological condition, when they are obtained with precision be obtained by means of a biopsy needle aspirate, a lavage as defined herein. The reason for this is that we have found Sample, Scrapings and Surgical incisions or other means that gene expression, properly measured-with requisite known in the art. US 2004/0225449 A1 Nov. 11, 2004
0110 Selected Panels that is both highly precise and reproducible in Samples taken from the same individual under the same conditions. We 0111 Steps in Selecting constituents in a Selected panel have similarly found that Such concentration measurements may include Searching publicly available medical literature are reproducible and precise in Samples that are repeatedly for RNA or proteins or sets of RNAS or proteins that directly tested. or indirectly vary with a particular biological condition. A Selected panel containing up to 100 constituents may be 0115 We commonly run a sample through a panel in Selected. According to the condition being examined, just a quadruplicate; that is, a Sample is divided into aliquots and Small Subset of the Selected panel constituents may be for each aliquot we measure concentrations of each con informative. Therefore, Selected panels may have as few as stituent in a selected panel. Over a total of 900 constituent two constituents, or as many as 1000 or more constituents. assays, with each assay conducted in quadruplicate, we It is possible that even a Single constituent may make up a found an average coefficient of variation, (standard devia Selected panel. In determining membership of the Selected tion/average) 100, of less than 1 percent among results for panel of genes, it is not necessary for the panel to be an each assay. This figure is a measure of what we call exhaustive selection. Rather it is desired to obtain from the “intra-assay variability”. We have also conducted assays on Selected panel an expression profile that discriminates con different occasions using the same Sample material. With 72 Sistently with respect to the targeted physiological or bio assays, resulting from concentration measurements of con logical condition. Moreover, a Selected panel is not neces Stituents in a panel of 24 members, and Such concentration Sarily Selected according to an expected profile of gene measurements determined on three different occasions over expression in cells that directly respond to a biological time, we found an average coefficient of variation of leSS effect. For example, gene expression associated with liver than 2 percent. We regard this as a measure of what we call metabolism may be analyzed in a blood sample. FIGS. 20 “inter-assay variability”. and 22 provide calibrated profiles of whole blood treated 0116 We have found it valuable in using the quadrupli with herbal agents using markers for liver metabolism. cate test results to identify and eliminate data points that are 0112 The number of constituents in a selected panel can statistical “outliers'; such data points are those that differ by vary. According to the examples provided below, Selected a percentage greater, for example, than 3% of the average of panels of up to 24-96 genes are Selected for evaluating all four values and that do not result from any Systematic expression levels. Although a Selected panel may be as large skew that is greater, for example, than 1%. Moreover, if as 100 constituents, it is desirable for a particular Selected more than one data point in a set of four is excluded by this panel to have no more than 24 constituents, more particu procedure, then all data for the relevant constituent is larly, less than 12 constituents. For example, Subsets of no discarded. more than 8 genes have been used that may be derived from 0117. As discussed in further detail below under “Gene a larger panel but which are Sufficiently informative to Expression”, we have also found it valuable to optimize the effectuate discrimination. The number of constituents in a efficiency of amplification for all constituents of a panel in Selected panel for which expression is monitored may vary a manner to achieve comparable amplification efficiencies widely depending on the context. For example, FIG. 1 (that is, amplification efficiencies that are Substantially simi describes data acquisition from in vitro cell culture and from lar as described below under “Gene Expression”) for all animal toxicology Studies, which includes expression of constituents, So that precise quantification of gene expres about 25 to 100 or more genes. In contrast, selection of Sion of all panel constituents may be determined consistently markers or Surrogate markers include, for example, three to on Successive occasions. In this manner, there may result 100 genes, preferably five to 50 or five to 25 genes to be data that is useful because it is precise and reliable. analyzed from Samples obtained in clinical Studies. In this manner markers or Surrogate markers having predictive 0118 What this approach means, among other things, is value for a medical condition, Such as a genetic predispo that by utilizing a relatively Small panel, and by controlling Sition, a response to therapeutic agent, an inflammatory experimental parameters for the whole panel, Such as Sam condition, or an infection, etc. can be identified and cumu pling, primer Selection, amplification efficiency and other latively larger populations can be obtained to refine the parameters, we create a panel that is uniquely informative. correlations. A health profile can then be generated for an This approach differs from prior art endeavors where speci individual Subject using a low volume blood Sample. The ficity is optimized only on a per-constituent basis and blood Sample can be analyzed for expression profile data of reaction conditions are not optimized for the panel as a about 100-500 genes, comprising markers or Surrogate whole. markers of a number of medical conditions (FIG. 1: right 0119) It is envisaged that any single biological condition panel). Selected panels of varying sizes may be utilized as may be described by a Signature panel having a Small necessary and Subsequent refinements in methodology may number of highly informative constituents providing a Sig lead to Selection of Subsets having Selected panels as large nature calibrated profile (also referred to as a fingerprint). as 15 genes or 12 genes or as Small as 6, 5, 4, 3 or 2 genes. Such a signature panel may have as few as two or more 0113) We have found that precision, including the con constituents. The presence of highly informative loci is cepts of repeatability and reproducibility, can be achieved in demonstrated in Several of the accompanying figures. For measurements for constituents in profile data Sets for example, FIG.11(a) Il-2, Il-4 and Il-5 are highly informa Selected panels wherein coefficients of variation are less than tive. Highly informative constituents in FIG. 21 include the 3 percent pro-inflammatory-interleukins. The Signature panel may provide a signature profile or fingerprint which is Sufficiently 0114. For example, we have found that we can measure robust to Serve as a Standard in describing a particular concentrations of constituents in Selected panels in a manner biological condition or an effect of a particular agent on a US 2004/0225449 A1 Nov. 11, 2004 biological condition For purposes of illustrating a Signature PCR is performed using amplification, reporting agents and panel, constituents of a Selected panel for measuring inflam instruments Such as those Supplied commercially by Applied mation have been provided that are informative with respect Biosystems (Foster City, Calif.). Given a defined efficiency to a particular biological condition. For example, we have of amplification of target transcripts, the point (e.g., cycle used a Selected panel for inflammation that has 6 constitu number) that signal from amplified target template is detect ents-Il-1C, Il-6, Il-8, Il-18, GMCSF and IFN-Y in FIG. able may be directly related to the amount of Specific 18(a)-(e) to determine the response of 5 subjects to varying message transcript in the measured Sample. Similarly, other concentrations of drugs. This group of constituents is a quantifiable Signals. Such as fluorescence, enzyme activity, Subset of a larger Selected panel of inflammation related disintegrations per minute, absorbance, etc., when correlated gene loci such as shown in FIG. 19a and FIG. 19b where the Inflammation Selected Panel includes Il-O, Il-B, Il-2, to a known concentration of target templates (e.g., a refer Il-3, Il-4, Il-6, Il-7, Il-8, Il-10, Il-12p40, Il-15, Il-15, Il-18, ence Standard curve) or normalized to a standard with GM-CSF, Ifn-gamma, TGF-B, cox-2, ICE, MMP-9, ICAM, limited variability can be used to quantify the number of TNF-C. and TNF-B. The subset of constituents were selected target templates in an unknown Sample. on the basis of the information Sought concerning the 0123. Although not limited to amplification methods, biological condition. quantitative gene expression techniques may utilize ampli 0120 Embodiments of the invention provide examples of fication of the target transcript. Alternatively or in combi numerous different Selected panels which may be used nation with amplification of the target transcript, amplifica Separately or together. These Selected panels include an tion of the reporter Signal may also be used. Amplification Inflammation Selected Panel (Table 1) a Cell Growth and of the target template may be accomplished by isothermic Differentiation Selected Panel (Table 2), a Liver Metabolism gene amplification Strategies, or by gene amplification by and Toxicity Selected Panel (Table 3). We have developed thermal cycling such as PCR. additional Selected panels including Skin Response Selected Panel (Table 4), Prostate Selected Panel (Table 5)(for mea 0.124. It is desirable to obtain a definable and reproduc Suring prostate health and disease), Vascular Selected Panel ible correlation between the amplified target or reporter and (Table 6)(for measuring condition of the vascular System the concentration of Starting templates. We have discovered and endothelial cells). It is a significant property of each of that this objective can be achieved by careful attention to, for these Selected panels that measurement of the Selected example, consistent primer-template ratioS and a strict panels constituents provides a measurement of the physi adherence to a narrow permissible level of amplification ological condition to which the Selected panel is targeted. efficiencies (for example 99.8 to 100% relative efficiency). Selected panels may also provide useful information con For example, in determining gene expression levels with cerning gene response outside the target condition. In these regard to a single Selected profile, it is necessary that all tables the left-hand column identifies the particular gene constituents of the panels maintain a similar and limited loci, and the right-hand column describes proteins expressed range of primer template ratios (for example, within a by these loci. However, as described in detail below, 10-fold range) and amplification efficiencies (within, for embodiments of the present invention may utilize, for example, less than 1%) to permit accurate and precise example, mRNA or protein expression products as constitu relative measurements for each constituent. We regard ents. While below we provide examples based primarily on amplification efficiencies as being "Substantially similar', the Inflammation Selected Panel and Subsets of it, the for the purposes of this description and the following claims, approaches Set forth herein are equally applicable to the if they differ by no more than approximately 10%. Prefer other Selected panels described above. Although provided as ably they should differ by less than approximately 2% and examples, the above Selected panels are not intended to be more preferably by less than approximately 1%. These limiting. constraints should be observed over the entire range of concentration levels to be measured associated with the 0121 Gene Expression relevant biological condition. In practice, we run tests to 0122) For measuring the amount of a particular RNA in assure that these conditions are Satisfied. For example, we a Sample, we have used methods known to one of ordinary typically design and manufacture a number primer-probe skill in the art to extract and quantify transcribed RNA from Sets, and determine experimentally which Set gives the best a Sample with respect to a constituent of a Selected panel performance. Even though primer-probe design and manu (See detailed protocols below.) Briefly, RNA is extracted facture can be enhanced using computer techniques known from a sample Such as a tissue, body fluid (see Example 11 in the art, and notwithstanding common practice, we still below), or culture medium in which a population of a Subject find that experimental validation is useful. Moreover, in the might be growing. For example, cells may be lysed and RNA course of experimental validation, we associate with the eluted in a Suitable Solution in which to conduct a DNAse Selected primer-probe combination a Set of features: reaction. First strand synthesis (see Example 10 below) may 0125 (i) The reverse primer should be complemen then be performed using a reverse transcriptase. Gene ampli tary to the coding DNA Strand; located acroSS an fication, more specifically quantitative PCR assays, can then intron-exonjunction, with not more than three bases be conducted and the gene of interest size calibrated against of the three-prime end of the reverse primer comple a marker such as 18S rRNA (Hirayama et al., Blood 92, mentary to the proximal exon. (If more than three 1998: 46-52). Samples are measured in multiple duplicates, bases are complementary, then it would tend to for example, 4 replicates. Relative quantitation of the mRNA competitively amplify genomic DNA.) is determined by the difference in threshhold cycles between the internal control and the gene of interest (see Example 12 0126 (ii) The primer probe should amplify cDNA of below). In an embodiment of the invention, quantitative less than 110 bases in length. US 2004/0225449 A1 Nov. 11, 2004
0127 (iii) The primer probe should not amplify conducting clinical trials or for characterizing a drug. The genomic DNA or transcripts or cDNA from related data may be transferred in networks via the World Wide but biologically irrelevant loci. Web, email, or an internet access Site, for example, or by hard copy So as to be collected and pooled from distant 0128. A suitable target of the selected primer probe is first geographic Sites Strand cDNA, which may be prepared, in one embodiment, according to Example 1 below. In Example 11 below, we 0133. The figures provided here are directed to RNA. illustrate use of the primer probe with the first strand cDNA However, methods herein may also be applied using proteins of Example 1 to permit measurement of constituents of a where Sensitive quantitative techniques, Such as an Enzyme Selected panel. Linked ImmunoSorbent Assay (ELISA), or amplification of nucleic acid aptamers that bind Specifically to gene expres 0129. It is envisaged that techniques in the art using Sion products, as detailed in our co-pending application, microfluidics for example and highly Sensitive markers will enable quantitation of RNA to occur directly from a single U.S. application Ser. No. 09/595,720, filed Jun. 16, 2000, cell or lysed cell. This may rely on amplification of a marker which is hereby incorporated by reference. These methods but may not require amplification of the transcripts them and others are available and well-known in the art for Selves. The amount of transcript measured for any particular measuring the amount of a protein constituent. locus is a data point or member of the first profile data Set 0134) Baseline Profile Data Sets for a particular Selected panel. 0.135 The analyses of samples from single individuals 0130. According to embodiments of the invention, a first and from large groups of individuals provide a library of profile data set is derived from the sample, the first profile profile data Sets relating to a particular panel or Series of data Set including a plurality of members, each member panels. These profile data Sets may be Stored as records in a being a quantitative measure of the amount of a RNA library for use as baseline profile data sets, or may them transcribed from a gene locus, the gene locus being a Selves describe a biological condition at the time and cir constituent in a panel of constituents. A first profile data Set cumstances of the particular Sample from which the profile may be obtained from a quantitative measure of the amount data Set is obtained, and be stored as records in a library for of a distinct RNA or protein corresponding to a gene locus. a particular biological condition for that particular Sample. Each member of the profile data set should be reproducible AS the term “baseline” Suggests, the Stored baseline profile within a range with respect to Similar Samples taken from the data Sets Serve as comparators for providing a calibrated subject under similar conditions. For example, the profile profile data set that is informative about a biological con data Sets may be reproducible within one order of magnitude dition or agent. Baseline profile data Sets may be Stored in with respect to Similar Samples taken from the Subject under libraries and classified in a number of cross-referential ways. Similar conditions. More particularly, the members may be One form of classification may rely on the characteristics of reproducible within 50%, more particularly reproducible the panels from which the data Sets are derived. Another within 20%, and sometimes even within 10% or less than form of classification may be by particular biological con within 3%. In addition, each member of the profile data set dition. The concept of biological condition encompasses any should be repeatable within a range with respect to multiple State in which a cell or population of cells may be found at assays of aliquots of the same Sample. For example, the any one time. This State may reflect geography of Samples, profile data Sets may be repeatable within one order of SeX of Subjects or any other discriminator. Some of the magnitude with respect to multiple assays of aliquots of the discriminators may overlap. The libraries may also be Same Sample. More particularly, the members may be accessed for records associated with a single Subject or repeatable within 50%, more particularly repeatable within particular clinical trial. The classification of baseline profile 20%, and Sometimes even within 10% or less than within data Sets may further be annotated with medical information 3%. about a particular Subject, a medical condition, a particular agent etc. 0131. In accordance with embodiments of the invention, a pattern of increasing, decreasing and no change in relative 0.136 The choice of a baseline profile data set for creating gene expression from each of a plurality of gene loci a calibrated profile data Set is related to the biological examined in the precision panel may constitute profile Sets condition to be evaluated, monitored, or predicted, as well that are informative with regards to a biological condition, as, the intended use of the calibrated panel, e.g., as to biological efficacy of an agent, treatment conditions or for monitor drug development, quality control or other uses. It comparison to populations. Patterns of this nature may be may be desirable to access baseline profile data Sets from the used to identify likely candidates for a drug trial, used in Same Subject for whom a first profile data Set is obtained or combination with other clinical indicators to be diagnostic or from different Subject at varying times, exposures to Stimuli, prognostic with respect to a biological condition or may be drugs or complex compounds, or may be derived from like used to guide the development of a pharmaceutical or or dissimilar populations. nutraceutical through manufacture, testing and marketing. 0.137 The profile data set may arise from the same Such profile data Sets may also be used to guide the subject for which the first data set is obtained, where the allopathic or naturpathic treatment of the particular biologi Sample is taken at a Separate or similar time, a different or cal condition that the profile data Set describes. Similar Site or in a different or similar physiological condi 0132) The numerical data obtained from quantitative tion. For example, FIG. 5 provides a protocol in which the gene expression and numerical data from gene expression Sample is taken before Stimulation or after Stimulation. The relative to a baseline profile data Set may also be Stored in profile data Set obtained from the unstimulated Sample may databases or digital Storage mediums and may be retrieved Serve as a baseline profile data Set for the Sample taken after for purposes including managing patient health care or for Stimulation, or it may describe a biological condition for a US 2004/0225449 A1 Nov. 11, 2004
given Subject or population of cells from which the Sample ventions, aging or exposure to environmental Stimuli or was obtained at a particular time and under particular toxins of the subject (FIG. 25). circumstances. The baseline data Set may also be derived 0.141. A use of a calibrated profile data set is to evaluate from a library containing profile data Sets of a population of a biological condition of a Subject. This may be for purposes Subjects having Some defining characteristic or biological of diagnosis or prognosis of a clinical disorder. It is desirable condition. The baseline profile data Set may also correspond to obtain a calibrated data Set that describes a State of health to Some ex vivo or in vitro properties associated with an in or alternatively a State of age or body mass or any condition vitro cell culture. The resultant calibrated profile data sets or State that an individual Subject might find themselves to may then be stored as a record in a database or library (FIG. be in. For example, the biological condition may relate to 6) along with or separate from the baseline profile database physical activity, conditioning or exercise, mental State, and optionally the first profile data Set although the first environmental factor Such as medication, diet, or geography profile data Set would normally become incorporated into a or exposure to radiation or environmental contamination or baseline profile data set under Suitable classification criteria. infectious agent, biological or environmental toxin. If health or conversely a clinical disorder is being evaluated, cali 0.138. Selected baseline profile data sets may be also be brated profiles data Sets may be used for monitoring change used as a Standard by which to judge manufacturing lots in in health Status by periodic or regular comparison of pro terms of efficacy, toxicity, etc. Where the effect of a thera files, the disorder may be a complex disease process possi peutic agent is being measured, the baseline data Set may bly involving multiple gene including inflammation, autoim correspond to gene expression profiles taken before admin mune disease, degenerative disease, allergy, Vascular istration of the agent. Where quality control for a newly disease, ischemia, developmental disease, hormonal condi manufactured product is being determined, the baseline data tions and infectious diseases. The clinical disorder may Set may correspond with a gold Standard for that product. further include arthritis, asthma, multiple Sclerosis and peri However, any Suitable normalization techniques may be menopausal changes. The biological condition may affect a employed. For example, an average baseline profile data Set System of a Subject including a respiratory, vascular, ner is obtained from authentic material of a naturally grown Vous, metabolic, urinary, reproductive, Structural and immu herbal nutraceutical and compared over time and over nological System or other metabolic State. The above different lots in order to demonstrate consistency, or lack of examples of a biological condition are given by way of consistency, in lots of compounds prepared for release. illustration and are not intended to be limiting. 013:9 Calibrated Data 0142. Similarly, calibrated profile data sets may be used 0140. A calibrated profile data set may be described as a to measure, monitor or predict the host response to an function of a member of a first profile data Set and a infectious agent for purposes of identifying the infectious corresponding member of a baseline profile data Set for a agent, assessing the duration of infection, the extent of given gene locus in a panel. For example, calibrated profile exposure or making therapeutic decisions. data Sets may be derived by calculating a ratio of the amount 0143. The evaluation of activity of an agent may require of RNA transcribed for a panel constituent in a cell sample a series of calibrated profiles. It is here shown that calibrated in an environmental including intervention Such as a thera profile data Sets may be used to describe the biological peutic treatment or at a particular time (first profile data Set) activity of an agent that may be a single compound or a with respect to the amount of RNA transcribed for the same complex compound Such as a nutraceutical or herbal. The panel constituent in a cell that differs in Some manner from agent may be assayed using indicator cells, eX Vivo cell the sample (baseline profile data set) (FIGS.5 and 6). Given populations or by in Vivo administration. These assays may the precision we have achieved in measurement of gene rely on a Series of Signature panels or enlarged panels for expression, described above in connection with "Selected different biological conditions. The resultant calibrated pro panels' and "gene amplification', we conclude that where files may then be used to infer likely in vivo activity from the differences occur in measurement under Such conditions, the in vitro Study. Insights into toxicity and mechanisms of differences are attributable to differences in biological con action can also be inferred from calibration profile data Sets. dition. Thus we have found that calibrated profile data sets For example, the herbal Echinacea is believed to have both are highly reproducible in Samples taken from the same immunostimulatory and anti-inflammatory properties individual under the same conditions. We have similarly although neither has been measured Systematically. We have found that calibrated profile data Sets are reproducible in provided a Systematic approach to investigate the biological Samples that are repeatedly tested. We have also found activities of these and other herbs. We investigated the repeated instances wherein calibrated profile data Sets alleged immunostimulatory properties of the herbs by com obtained when Samples from a Subject are exposed ex vivo paring the effect of treating the indicator cell line THP-1 or to a compound are comparable to calibrated profile data peripheral blood cells with the agent to untreated cells. from a Sample that has been exposed to a Sample in vivo Untreated cells include LPS stimulated untreated cells. (FIG. 14, FIG. 16(a), (b), and FIGS. 29(a) and 29(b)). We Untreated cells were used as a baseline profile data Set to have also found, importantly, that an indicator cell line measure the difference in gene expression between a base treated with an agent can in many cases provide calibrated line profile data Set and the experimental treatment with the profile data Sets comparable to those obtained from in vivo compound. Baseline profile data Sets included a single or ex vivo populations of cells (FIG. 15). Moreover, we Sample or an average value from a Series of experiments. have found that administering a Sample from a Subject onto The resultant calibrated profile data sets could then be indicator cells can provide informative calibrated profile compared with a library of calibrated profile data sets for a data Sets with respect to the biological condition of the particular herb or/and libraries associated with different Subject including the health, disease States, therapeutic inter agents or conditions. US 2004/0225449 A1 Nov. 11, 2004
0144. From the information obtained about a previously organized So as to provide an output optionally correspond undescribed agent, a Signature panel may be derived option ing to a graphical representation of a calibrated data Set. ally together with a Signature profile to Serve as a gold 0151. For example, a distinct sample derived from a Standard for testing other batches of the same agent. subject being at least one of RNA or protein may be denoted 0145 Calculation of Calibrated Profile Data Sets and as P. The first profile data set consists of M, where Mi is a Computational Aids quantitative measure of a distinct RNA or protein constitu 0146 The function relating the baseline and profile data ent. The record Riis a ratio of M and P and may be annotated Sets is, in an embodiment of the invention, a ratio expressed with additional data on the Subject relating to for example, as a logarithm. The calibrated profile data Set may be age, diet, ethnicity, gender, geographic location, medical expressed in a spreadsheet or represented graphically for disorder, mental disorder, medication, physical activity, example, in a bar chart or tabular form but may also be body mass and environmental exposure. Moreover, data expressed in a three dimensional representation. The con handling may further include accessing data from a Second Stituent may be itemized on the X-axis and the logarithmic condition database which may contain additional medical Scale may be on the y-axis. Members of a calibrated data Set data not presently held with the calibrated profile data Sets. may be expressed as a positive value representing a relative In this context, data access may be via a computer network. enhancement of gene expression or as a negative value 0152 The above described data storage on a computer representing a relative reduction in gene expression with may provide the information in a form that can be accessed respect to the baseline. by a user. Accordingly, the user may load the information 0147 Each member of the calibrated profile data set onto a Second acceSS Site including downloading the infor should be reproducible within a range with respect to Similar mation. However, access may be restricted to users having Samples taken from the Subject under Similar conditions. For a password or other Security device So as to protect the example, the calibrated profile data Sets may be reproducible medical records contained within. A feature of this embodi within one order of magnitude with respect to Similar ment of the invention is the ability of a user to add new or Samples taken from the Subject under Similar conditions. annotated records to the data Set So the records become part More particularly, the members may be reproducible within of the biological information. 50%, more particularly reproducible within 20%, and some 0153. The graphical representation of calibrated profile times even 10%. In accordance with embodiments of the data Sets pertaining to a product Such as a drug provides an invention, a pattern of increasing, decreasing and no change opportunity for Standardizing a product by means of the in relative gene expression from each of a plurality of gene calibrated profile, more particularly a Signature profile. The loci examined in the precision panel may be used to prepare profile may be used as a feature with which to demonstrate a calibrated profile Set that is informative with regards to a relative efficacy, differences in mechanisms of actions, etc. biological condition, biological efficacy of an agent treat compared to other drugs approved for Similar or different ment conditions or for comparison to populations. Patterns SCS. of this nature may be used to identify likely candidates for a drug trial, used in combination with other clinical indica 0154) The various embodiments of the invention may be tors to be diagnostic or prognostic with respect to a biologi also implemented as a computer program product for use cal condition or may be used to guide the development of a with a computer System. The product may include program pharmaceutical or nutraceutical through manufacture, test code for deriving a first profile data Set and for producing ing and marketing. calibrated profiles. Such implementation may include a Series of computer instructions fixed either on a tangible 0.148. The numerical data obtained from quantitative medium, Such as a computer readable medium (for example, gene expression and numerical data from calibrated gene a diskette, CD-ROM, ROM, or fixed disk), or transmittable expression relative to a baseline profile data Set may be to a computer System via a modem or other interface device, Stored in databases or digital Storage mediums and may Such as a communications adapter coupled to a network. The retrieved for purposes including managing patient health network coupling may be for example, over optical or wired care or for conducting clinical trials or for characterizing a communications lines or via wireless techniques (for drug. The data may be transferred in networks via the World example, microwave, infrared or other transmission tech Wide Web, email, or internet access site for example or by niques) or Some combination of these. The Series of com hard copy So as to be collected and pooled from distant puter instructions preferably embodies all or part of the geographic sites (FIG. 8). functionality previously described herein with respect to the 0149. In an embodiment of the present invention, a System. Those skilled in the art should appreciate that Such descriptive record is Stored in a single database or multiple computer instructions can be written in a number of pro databases where the Stored data includes the raw gene gramming languages for use with many computer architec expression data (first profile data Set) prior to transformation tures or operating Systems. Furthermore, Such instructions by use of a baseline profile data Set, as well as a record of may be stored in any memory device, Such as Semiconduc the baseline profile data set used to generate the calibrated tor, magnetic, optical or other memory devices, and may be profile data Set including for example, annotations regarding transmitted using any communications technology, Such as whether the baseline profile data set is derived from a optical, infrared, microwave, or other transmission technolo particular signature panel and any other annotation that gies. It is expected that Such a computer program product may be distributed as a removable medium with accompa facilitates interpretation and use of the data. nying printed or electronic documentation (for example, 0150. Because the data is in a universal format, data Shrink wrapped Software), preloaded with a computer Sys handling may readily be done with a computer. The data is tem (for example, on system ROM or fixed disk), or dis US 2004/0225449 A1 Nov. 11, 2004
tributed from a server or electronic bulletin board over a show how the effect of methotrexate and meclofenamate network (for example, the Internet or World Wide Web). In generates Similar calibrated profile data Sets where the addition, a computer System is further provided including baseline is LPS treated blood. In contrast, methylpredniso derivative modules for deriving a first data Set and a cali lone has a substantially different effect from the other two bration profile data Set. compounds. A similar type of analysis can be performed with complex mixtures, as illustrated in FIG. 21, in which O155 Clinical Trials the calibrated profiles obtained when Echinacea, Arnica and 0156 The use of calibrated profile data sets for perform Siberian Ginseng applied to LPS stimulated blood ex vivo ing clinical trials is illustrated in FIG. 10 using the above are compared. In this example, all three agents appear to act described methods and procedures for running a clinical trial differently from each other with respect to a Sample from a or managing patient care. Moreover, Standardization Single Subject. Similar analyses can be used to compare between laboratories may be achieved by using a particular compounds with unknown targets or activities or metabolic indicator cell line such as THP-1 which is stimulated by a patterns to compounds, complex or simple, with known or known Stimulator Such as lipopolysaccharide So that result pre-determined profiles. ant profile acts as a measure that the laboratory is performing 0159. The above methods and procedures may be utilized the protocol correctly. Of course this is one Single example, in the design and running of clinical trials or as a Supple and other cells lines, tissues, or biological Samples or mental tool. Moreover, the above methods and procedures combinations of the foregoing may be used as Standards. may be used to monitor the patients health as well as the O157 A further embodiment of the invention provides a patient's responsiveness to an agent before during and after method for patient Selection for augmenting clinical trials. the clinical trial. This includes monitoring whether multiple Clinical trials in which candidate Subjects are included or agents interfere with each other, act Synergistically or addi excluded according to a predetermined optimum calibrated tively or are toxic or neural with respect to each other. This profile for a given biological condition can result in more type of information is very important as individuals take an precise monitoring than would be otherwise possible. It can increasing number of medications. also result in a greater efficiency in clinical trial design 0160 Similarly, the methods and procedures described becauSe unsuitable patients that have for example compli above may be used to manage patient care for an individual cating factors or conditions can be Screened out. The cali or a population. Such methods and procedures may also be brated profile data will also enhance the “signal to noise” by used to develop a regional or global research network that removing non-responders from clinical Studies. The basic uses calibrated profile data sets and the resulting databases Structure of a clinical trial design using gene expression to conduct research or trials. profiling may follow any of Several formats. These include 0.161 Both the calibration profile data sets in graphical testing body fluid from a candidate patient in the trial ex vivo form and the associated databases together with information against a new therapeutic agent and analyzing the calibrated extracted from both are commodities that can be sold profiles with respect to an agent-treated and placebo-treated together or Separately for a variety of purposes. For Samples using a predetermined Selected panel and evaluating example, graphic representations of calibration profile data whether the candidate patient would be likely to respond Sets may provide a description of a product with respect to without adverse effects to the composition being tested. In its activity that may be used to promote the product. Alter Selected indications, profile data obtained from in vitro cell natively, the graphical form of the calibrated profile data Sets cultures or organ cultures may be desired where the cell and access to baseline profile databaseS provide a means for originates from a target Subject or from another Subject or manufacturers to test discrete batches of product against a from an established cell line, or from a cell Samples removed gold Standard. from the target Subject where the cell Samples may be obtained from any body fluid including a blood, urine, 0162 The data may be used strategically for design of Semen, amniotic, or a cerebroSpinal fluid Sample, or from a clinical trials. It may also be useful for physicians practicing Scraping from mucosal membranes Such as from the buccal at remote sites to offer personalized healthcare to a patient. cavity, the eye, nose, vagina or by means of a biopsy Accordingly, the physician may set up personalized data including epithelial, liver, Sternum marrow, testicular, or bases for calibrated profile data Sets prior to and after from tumor tissue removed Surgically from a tumor at any treatment of a particular condition. New data on the Subject location. The above-described Sources of Samples are appli could be added to the personalized database at each visit to cable to any medical use in which calibrated profile data Sets the doctor. The data may be generated at remote sites by the are desired. use of kits that permit a physician to obtain a first profile data Set on a Sample from a patient. For remote users to access the 0158. In vitro dosage and toxicity studies using calibrated Site, it is envisaged that Secured access to the global network profile data Sets obtained from indicator cell lines or Samples containing libraries of baseline profile data Sets and cali of the patient tested eX Vivo may provide useful information brated profile data Sets, classified by particular criteria and prior to initiation of the clinical trial and may significantly representing data from larger populations than a Single reduce the cost and time of a clinical trial while increasing individual, would be necessary. The access to the global the likelihood of identifying the presence of beneficial database may be password protected thereby protecting the effect(s). In particular, the dose may be optimized on an database from corrupted records and Safeguarding personal individualized basis to maximize the impact on therapeutic medical data. The graphical form provided by the calibrated outcome. For example, FIG. 12 shows how ex vivo blood data Sets may be used to create catalogs of compounds in a cells respond to the stimulatory effect of LPS and the pharmacopiae complete with toxic effects that might arise Subsequent treatment with an anti-inflammatory drug (meth for particular individuals as well as other types of drug otrexate, meclofenamate or methylprednisolone). The data interactions. US 2004/0225449 A1 Nov. 11, 2004
0163 Access to the global database may include the Sample by methods known to one or ordinary skill in the art option to load Selected data onto a Second access site. This (see, for example, the Lyse-N-GoTM reagent, Pierce Chem. proceSS may include downloading the information to what Co., Rockford, Ill.). Samples are analyzed by QPCR accord ever Site is desired by the user and could include Securing ing to a quantitative replicative procedure, (for example, hard copies of information. It is desirable to control how and quantitative polymerase chain reaction procedure. (QPCR)) what data is offloaded or copied to maintain the integrity of (see, for example, Gibson, U. 1996 Genome Res. 6:995 the database. It is envisaged that while a global network of 1001, and references cited therein). Total RNA was assessed clinical data would be an informational resource, it would using universal primers. Toxicity of the agent for cells can have utility in conducting research that may include epide be measured in untreated cells by Vital Stain uptake, rate of miological Studies and Studies concerning the mechanism of DNA synthesis (autoradiography of labeled nucleic com action of an agent, as well as Studies concerning the nature pared to cells stained), stain by DNA-specific eyes of interpersonal variability as determined by calibrated (Hoechst), etc. Mechanistic profiles can be determined by profile data Sets. analysis of the identifies of de novo up- or down-regulated genes. Further, in the presence of a therapeutic agent, Some 0164. Examples of Medical Uses genes are not expressed or differentially expressed, indicat 0165 (a) Early detection of infectious diseases: Markers ing potential efficacy of the therapeutic agent in Suppressing or Surrogate markers from mice may be obtained for mea the effects of stimulation by the LPS. For example, in FIG. Suring gene expression in humans that indicate early or 21, levels of ICE that are somewhat stimulated in the immediate response to infection, for example, to a virus Such presence of LPS+Echinacea are Substantially depressed by as hepatitis virus, or to a bacterium Such as Mycobacterium LPS+Arnica relative to LPS stimulated cells absent agent. tuberculosis (the etiologic agent of tuberculosis) (see FIG. Levels of HSP 70 which are depressed in the presence of 4). Candidate genes are identified and changes in expression LPS+Echinacea are Substantially Stimulated in the presence of those genes in the presence of a challenge provide a Set of LPS+Arnica, and LPS+Siberian Ginseng relative to LPS of markers. The Set of markers can combine markers Stimulated cells absent the addition of an agent. Levels of encoded by the genome of the Subject and one more dis IL-112p40 which are slightly increased in the presence of tinctive markers encoded by the genome of the infectious LPS+Echinacea are Substantially depressed in the presence agent. For example, changes in expression of an immediate of LPS+Arnica and LPS+Siberian Ginseng relative to LPS early gene of a Virus, e.g. a gene encoding an enzyme of Viral stimulation. Similarly, FIG. 16 shows a much enhanced replication, and a host gene Such as the gene for any or all reduction of gene expression in whole blood for IL-1C, of IL-2, IL-4 and IL-5, may comprise markers or Surrogate Il-1 B, Il-7, Il-10, IL-IL-15, IFN-Y, TGF-B, TNF-B cox-2, and markers for a medical condition capable of detecting that ICAM in the presence of prednisolone +LPS when com condition prior to the onset of medical Symptoms. This pared to arnica +LPS or nothing +LPS. method may afford earlier detection of an infection than is 0168 (c) Quantitation of gene expression in a blood cell possible using current diagnostic techniques. to predict toxicity in another tissue or organ. 0166 (b) Toxicity profiles and mechanistic profiles 0169. Leukocytes, for example, may be obtained from a obtained from an in Vitro assay and in Vivo assayS. Toxicity blood Sample of a Subject, for the purpose of assessing the and mechanistic information arising from the administration appearance of a pathological condition in another organ, for of a compound to a population of cells may be monitored example, the liver. A profile data Set is obtained of genes using calibrated profile data Sets. The following is an expressed in the leukocytes, for example, genes encoding a example of an experimental protocol for obtaining this Set of lymphokines and cytokines. The data Set is compared information. Firstly, an experimental group is established: to that of the database, to examine correlations, for example (1) control cells maintained without therapeutic agent and to other Subjects, and to the Subject prior to administration without stimulus; (2) cells treated with therapeutic agent but of a therapeutic agent. without stimulus; (3) cells without therapeutic agent but with Stimulus, (4) sample with therapeutic agent and with 0170 By this method, a correlation can be drawn Stimulus. The population of cells can be Selected from between, for example, administration of acetaminophen primary cell cultures prepared in culture plates using meth (Tylenol) and Sensitivity to this therapeutic agent and mani ods well established in the art; or mature differentiated cell fested by liver damage. An early prediction of therapeutic preparation from whole blood or isolated monocytes from agent Sensitivity, detected prior to the onset of actual damage the target organism. to the liver, may be clinically available so that the subject receives no further administration of acetaminophen. The 0167 The cells are stimulated so as to present a targeted database may be used to detect a correlation or correlations physiological condition by pretreatment with LPS purified prior to the onset of traditional medical assessments, Such as from a Gram-negative bacterium (a variety of LPS prepa increase in bilirubin level or other indication of liver pathol rations from pathogenic bacteria, for example, from Salmo Ogy. nella typhimurium and from Escherichia coli O1157:H7, are available from Sigma, St. Louis, Mo.). The therapeutic agent 0171 (d) Calibrated profiles from blood cells for prog administered to the cell Samples in this example is an nosis of Severity and prediction of adverse reactions in inhibitor of an enzyme known to be key in disease etiology, treatment of an autoimmune disease. namely an inhibitor of a protease or a nucleic acid poly 0172 The probability and timing of onset of symptoms of merase. Following treatment by addition of the therapeutic an autoimmune disease, for example, rheumatoid arthritis, agent and further incubation for four to Six hours, Samples may be monitored by appearance of expression of markers of the cells are harvested and analyzed for gene expression. or Surrogate markers as determined by the methods of gene Nucleic acid, specifically mRNA, can be prepared from the expression profiling of markers or Surrogate markers and US 2004/0225449 A1 Nov. 11, 2004 comparison to a profile database as described above. Thus an provides a quick and effective way to determine which drug, indication of onset may be obtained, and advance manage chosen from within a single class of drugs that all may be ment by utilization of preventive measures to forestall onset, used to treat a particular condition, may be most effective for can be taken. Further, the user may choose a set of potential a given Subject. Alternatively, an agent may be tested on an therapeutic agents, and assess for a given agent, the prob indicator cell line that can provide a quantitative measure of ability that a Subject will present an adverse reaction if given therapeutic performance in a class of individuals. a full course of treatment, prior to that full course. For example, using embodiments of the invention, a Single dose 0178 FIG. 2 illustrates how calibrated profile data sets or a few doses of the agent methotrexate may be adminis may assist in Screening a library of candidate compounds to tered to a Subject having arthritis and in need of a therapeutic discover candidate drugs. Starting with for example, 500 agent. If the gene expression profile data Set of the Subject candidate drugs, these can be tested in indicator cells or eX in response to the short course of methotrexate correlates Vivo body fluid or tissues against Signature panels for in Vitro with data Sets from Subjects having adverse reactions to this toxicology or metabolic indicators. The figure illustrates the agent, then administration of a full course of methotrexate is large number of compounds that entered in late Stages in the counterindicated. Conversely, if the gene expression profile development proceSS only to ultimately be rejected due to data set correlates with those of subjects who have adverse biological interactions. Use of calibrated profile data responded positively to administration of a course of meth Sets may in many instances more readily identify likely otrexate treatment, then this therapeutic agent can be admin Successful candidates and thereby reduce the expense and istered to the subject with much lower probability of adverse untoward effects of animal and human experimentation for reaction. compounds that could have been predicted to fail. 0173 Discussion of Figures 0179 FIG. 3 illustrates how a compound may be admin 0.174 FIGS. 1-4 illustrate some of the applications of istered to an experimental animal Such as a mouse or to an calibrated profile data sets. In FIG. 1, three possible sce indicator cell line. The in vivo or ex vivo or indicator cell narios are provided. Firstly, a candidate therapeutic agent sample may further be treated with a stimulus. The result of may be tested to determine its molecular pharmacology and both the compound and the Stimulus may then be detected, toxicology profiles. The test might include obtaining cali for example, using Signature profiles for toxicity or for brated profile data Sets for a Series of Selected panels Selected mechanism to compare the effect of no drug +/-Stimulus or on the basis of what activity is predicted for the drug. The +/-drug and no stimulus. Both in vitro (left panel of FIG. 3) population of cells exposed to the agent may be the result of and in vivo (right panel of FIG. 3) studies can be used to in Vivo administration as depicted by the mouse or direct evaluate the effect of a compound (drug, nutraceutical, exposure in Vitro where the cells may be an indicator cell environmental stimuli, etc.). The right hand panel also line or an ex vivo sample from the Subject. The result of the illustrates the specific embodiment of an “in vitro clinical Screen is the identification of more effective drug candidates trial', that is, treatment of cells obtained from a Subject and for testing in human Subjects. treated with a compound (with or without a stimulus) in vitro (or ex vivo) in order to predict the outcome of similar 0175. The second scenario in FIG. 1 is the use of treatment of the subject in vivo (see FIG. 15 for a specific calibrated profile data Sets to identify a Suitable clinical example). The output from both panels is described as population for Screening a potential therapeutic agent. Both toxicity and mechanistic profiles. Either experimental course demonstration of lack of toxicity and demonstration of may be used to both evaluate potential toxicity, e.g., using clinical efficacy require certain assumptions about the clini the toxicity, or liver metabolism Selected panels, and to cal population. The calibrated profile data Sets provide a determine or confirm likely mechanism of action by a means for establishing those assumptions with respect to the critical Selection of a gene panel(s) that illustrates and biological condition of the individuals selected for the differentiates molecular mechanisms of action (see FIG. 12 clinical trials. for a specific example). These are merely examples, and 0176) The third scenario in FIG. 1 involves the practice other Selected panels may be employed to evaluate or of individualized medicine, which may include creating an characterize other biological effects or conditions. FIG. 4 archive of calibrate profile data Sets on the individual in a illustrates a bioassay in which cells are removed from the State of health Such that changes can be identified using Subject and tested eX Vivo with the addition of a compound Signature panels So as to permit evaluation, prognosis, or and also a challenge or Stimulus. The eX Vivo effect of diagnosis of a particular condition. Moreover, Stored infor Stimulus and then drug on whole blood taken from a human mation about the patient in the form of calibrated profile data subject is shown in FIG. 12 in which the stimulus is Sets permits Selecting one of a group of possible therapeutic lipopolysaccharide (an inflammatory agent) while the drug agents most likely to be effective for the patient, optimizing is any of methotrexate, meclofenamate or methylpredniso dosage of drug, and detecting adverse effects that might arise lone using a Signature panel for inflammation. Methylpred through drug-drug interactions before Symptoms arise. Use nisolone, a drug commonly used in the treatment of acute exacerbations of COPD as well as in the chronic manage of calibrated profile data Sets may provide more efficient and ment of this disease, is considered to be a potent by cost-effective health care management. non-specific anti-inflammatory agent. However, as demon 0177. The novel approach described above for evaluating Strated in FIG. 22, its effects on gene expression are a biological condition of a Subject may be applied to an eX dependent on the Stimulus. While there are general qualita Vivo or in vitro assay for measuring the effect of an agent on tive Similarities between the effects on gene expression a biological condition as illustrated in FIGS. 2-4. A sample acroSS these three Stimuli, there are both quantitative and from the patient may measured directly eX Vivo or tested eX qualitative differences that may be important in understand Vivo against an agent to predict an effect in the patient. This ing when glucocorticoid intervention is warranted. US 2004/0225449 A1 Nov. 11, 2004
0180 According to embodiments of the invention, an mechanism of action, and to compare a single profile to a indicator cell population is used to measure quantitative collection of Signature, calibrated Selected profiles. gene expression the effect of an agent or a biological Sample 0185. Use of a database in accordance with an embodi may influence the choice of which indicator cell line will be ment of the present invention is illustrated in FIG. 8. FIG. most informative. For example, a cloned cell line Such as 8 illustrates a data profile set from the database. Entries for THP-1 or a primary cell population (peripheral mononuclear input include a name, an Experimental Type, and whether cells) may provide information that is comparable to that the entry is a New Reference; Cell/Tissue/Species and obtained from a body sample directly (see FIG. 15). The whether these are new; Therapeutic agent (compound), normal State of gene expression may range from Zero or few Dose, and additional parameters and whether the therapeutic transcripts to 10 or more transcripts. agent is new. Observations are recorded according to the 0181 Similarly, an agent may be evaluated for its effect identity of a Gene (New Gene) and a Protein (New Protein). on any population of cells, either in Vivo, eX Vivo or in vitro, The Stimulus or other Treatment, if any, and the Dose are by administering the agent and then determining a calibrate entered. Gene (and/or Protein) Expression, Expression profile data Set for those cells under the Selected conditions. Value, Expression Units if appropriate and Expression Time Examples of this approach are provided in FIGS. 10-16 and are shown. The figure Specifically illustrates the range of 18. FIG. 18 further provides calibrated profile data sets for applicable fields of investigation from complex natural different concentrations of a single agent showing that the products to clinical trails in humans, linkage to traditional transcription of Selected constituents vary with dose and forms of measurement and evaluation Such as literature therefore the anticipated effectiveness with respect to the citations, clinical indicators and traditional pharmacokinetic biological condition. measurements. Expert analysis of the Selected profile data contained in the database may then be used to guide product 0182. The above description of determining a biological development and marketing, or used to improve the clinical condition is exemplified as follows: the action of a pharma decision making concerning the health of a single individual ceutical or nutraceutical is measured with respect to its or population of individuals. anti-inflammatory properties. The measurement of the effect may be established using a Selected panel of constituent 0186 One form of record may provide information about gene loci for example, an inflammation Selected panel, a Subject or agent with respect to identity, medical history including, Interleukin 1 alpha (IL-1C) or Tumor Necrosis including traditional pharmaceutical/medical data, clinical Factor alpha (TNF-C). The anti-inflammatory effect may indications as determined from literature data, reference to first be established by treating indicator cells or Sample cells additional types of analysis in the database, etc. ex vivo with a known inflammation inducers (for example, 0187 FIG. 9 shows an embodiment of the present inven lipopolysaccharide or other mitogens) followed by treatment tion in which profile data is evaluated using data from a with the experimental agent or condition expected to affect database that is remotely accessed over a network. Using the the expression from the appropriate gene loci. Accordingly, approach of this figure data may be derived at one or more a baseline profile data Set may be established in this case as locations (Such as location 1 shown here), compared using the gene expression for a particular panel of constituents information retrieved over communication path 1109 from a resulting in the presence of the inflammation inducer. The central database at location 2, and the result of the compari addition of a potential anti-inflammatory agent results in a Son may be used to affect, for example, the course of change relative to the baseline. This approach is illustrated treatment of an individual or population. The communica for example in FIG. 12. Methylprednisolone has a substan tion path 1109 between location 1 and location 2 is two-way, tial down regulation effect on IL-2 in blood cells stimulated So that information resulting from determinations made at ex vivo with LPS where the baseline data set is LPS location 1 may delivered over the path 1109 to update the Stimulated cells. In this case the effect is shown as negative. database 1108. The consequence is an iterative process In contrast, as shown in FIG. 16b, IL-2 appears to be whereby the information from database is used in a deter upregulated in whole blood not previously exposed to LPS, mination that may affect the course of treatment, evaluation, where the baseline data Set is unstimulated cells. These or development, and the results of the determination become results are consistent with the observation that methylpred part of the database. In a first location, as in FIG. 5, from a nisolone Stimulates IL-2 production. tissue Sample procured in proceSS 1101, there are derived 0183 The determination of the biological condition of a multiple RNA species pursuant to process 1102, and then in Subject may include measuring and Storing additional data process 1103, profile data are quantified to produce a profile about the Subject. For example, if the Subject is a human or data Set that is pertinent to the tissue Sample obtained in mammalian patient, additional clinical indicators may be process 1101. In order to evaluate the profile data set, in determined from blood chemistry, urinalysis, X-ray, other process 1104 information is retrieved from database 1108, chemical assays and physical or Sociological findings. which is located in a Second location. In fact the database may be in communication with a large number of locations, 0184 FIG. 7 illustrates how the accumulation of cali each of which is generating profile data that must be brated profile data Sets may improve the predictive power of evaluated. The retrieval of information from the database is the database and thereby increase its value in generating accomplished over a communication path 1109, which may information about a biological condition or agent. The figure include a network Such as the Internet, in a manner known indicates the use of the database in terms of its power, for in the art. Once information has been obtained from the example, to predict the course of a therapeutic intervention database 1108, the information is used in evaluating the or follow the course of an individual Subject compared to a quantified profile data in process 1105, with the result in population. Information from the database may be used to process 1106 that the medical condition of the subject may predict a likely mechanism of metabolism or molecular be assessed. In process 1107, the database 1108 is updated US 2004/0225449 A1 Nov. 11, 2004 over the communication path 1109 to reflect the profile data blood cells, and 1 mL was removed from each tube (using that have been quantified in process 1103. In this manner the a micropipettor with barrier tip), and transferred to a 2 mL database 1108 may be updated to reflect the profile data “dolphin' microfuge tube (Costar #3213). obtained over all locations, and each location has the benefit of the data obtained from all of the locations. While, for 0193 The samples were then centrifuged for 5 min at simplicity, all of the processes in FIG. 9 are shown as taking 500xg, ambient temperature (IEC centrifuge or equivalent, place at location 1, Some or even all of the processes may be in microfuge tube adapters in Swinging bucket), and as much implemented elsewhere, for example location 2, or in mul Serum from each tube was removed as possible and dis tiple locations. At location 2, associated with the database, carded. Cell pellets were placed on ice; and RNA extracted for example, may be a Server that is used for hosting these as Soon as possible using an Ambion RNAqueous kit processes, including evaluation of the quantified profile data. 0194 (b) Amplification Strategies. EXAMPLES 0.195 Specific RNAS are amplified using message spe cific primers or random primers. The Specific primers are Example 1 Synthesized from data obtained from public databases (e.g., Unigene, National Center for Biotechnology Information, 0188 (a) Use of whole blood for ex vivo assessment of National Library of Medicine, Bethesda, Md.), including a biological condition affected by an agent. information from genomic and cDNA libraries obtained 0189 Human blood is obtained by venipuncture and from humans and other animals. Primers are chosen to prepared for assay by aliquotting Samples for baseline, no preferentially amplify from specific RNAS obtained from the Stimulus, and Stimulus with Sufficient volume for at least test or indicator samples, see, for example, RT PCR, Chapter three time points. Typical Stimuli include lipopolysaccharide 15 in RNA Methodologies, A laboratory guide for isolation (LPS), phytohemagglutinin (PHA) and heat-killed Staphy and characterization, 2nd edition, 1998, Robert E. Farrell, lococci (HKS) or carrageenan and may be used individually Jr., Ed., Academic Press; or Chapter 22 pp.143-151, RNA (typically) or in combination. The aliquots of heparinized, isolation and characterization protocols, Methods in whole blood are mixed without stimulus and held at 37 C. molecular biology, Volume 86, 1998, R. Rapley and D. L. in an atmosphere of 5% CO for 30 minutes. Stimulus is Manning Eds., Human Press, or 14 in Statistical refinement added at varying concentrations, mixed and held loosely of primer design parameters, Chapter 5, pp.55-72, PCR capped at 37° C. for 30 min. Additional test compounds may applications: protocols for functional genomics, M. A. Innis, be added at this point and held for varying times depending D. H. Gelfand and J. J. Sninsky, Eds., 1999, Academic on the expected pharmacokinetics of the test compound. At Press). Amplifications are carried out in either isothermic defined times, cells are collected by centrifugation, the conditions or using a thermal cycler (for example, a ABI plasma removed and RNA extracted by various standard 9600 or 9700 or 7700 obtained from Applied Biosystems, CS. Foster City, Calif.; see Nucleic acid detection methods, pp. 1-24, in Molecular methods for virus detection, D. L. 0190. Nucleic acids, RNA and or DNA are purified from Wiedbrauk and D. H., Farkas, Eds., 1995, Academic Press). cells, tissueS or fluids of the test population or indicator cell Amplified nucleic acids are detected using fluorescent lines. RNA is preferentially obtained from the nucleic acid tagged detection primers (see, for example, Taqman PCR mix using a variety of Standard procedures (or RNA Isola Reagent Kit, Protocol, part number 402823 revision A, tion Strategies, pp.55-104, in RNA Methodologies, A labo 1996, Applied Biosystems, Foster City Calif.) that are iden ratory guide for isolation and characterization, 2nd edition, tified and Synthesized from publicly known databases as 1998, Robert E. Farrell, Jr., Ed., Academic Press); in the described for the amplification primers. In the present case, present use using a filter-based RNA isolation System from amplified DNA is detected and quantified using the ABI Ambion (RNAqueousTM, Phenol-free Total RNA Isolation Prism 7700 Sequence Detection System obtained from Kit, Catalog #1912, version 9908; Austin, Tex.). Applied Biosystems (Foster City, Calif.). Amounts of spe 0191 In accordance with one procedure, the whole blood cific RNAS contained in the test sample or obtained from the assay for Selected profiles determination was carried out as indicator cell lines can be related to the relative quantity of follows: Human whole blood was drawn into 10 mL. Vacu fluorescence observed (see for example, Advances in quan tainer tubes with Sodium Heparin. Blood samples were titative PCR technology: 5' nuclease assays, Y. S. Lie and C. mixed by gently inverting tubes 4-5 times. The blood was J. Petropolus, Current Opinion in Biotechnology, 1998, used within 10-15 minutes of draw. In the experiments, 9:43-48, or Rapid thermal cycling and PCR kinetics, pp. blood was diluted 2-fold, i.e. per Sample per time point, 0.6 211-229, chapter 14 in PCR applications: protocols for mL whole blood +0.6 mL stimulus. The assay medium was functional genomics, M. A. Innis, D. H. Gelfand and J. J. prepared and the Stimulus added as appropriate. Sninsky, Eds., 1999, Academic Press. 0192 Aquantity (0.6 mL) of whole blood was then added 0196. As a particular implementation of the approach into each 12x75 mm polypropylene tube. 0.6 mL of 2xLPS described here, we describe in detail a procedure for Syn (from E. coli serotye 0127:B8, Sigmai L3880 or serotype thesis of first strand cDNA for use in PCR. This procedure 055, Sigma #LA005, 10 ng/ml, Subject to change in different can be used for both whole blood RNA and RNA extracted lots) into LPS tubes was added. Next, 0.6 mL assay medium from cultured cells (i.e. THP-1 cells). was added to the “control” tubes with duplicate tubes for each condition. The caps were closed tightly. The tubes were 0197) Materials inverted 2-3 times to mix Samples. Caps were loosened to 0198 (1) Applied Biosystems TAQMAN Reverse Tran first stop and the tubes incubated (G) 37 C., 5% CO for 6 scription Reagents Kit (P/N 808-0234). Kit Components: hours. At 6 hours, Samples were gently mixed to resuspend 10x TaqMan RT Buffer, 25 mM Magnesium chloride, deox US 2004/0225449 A1 Nov. 11, 2004
yNTPs mixture, Random Hexamers, RNase Inhibitor, Mul profile with respect to Some constituents to a change of 10' tiScribe Reverse Transcriptase (50 U/mL) (2) RNase/DNase (10E13) in gene expression of one constituent (indeed the free water (DEPC Treated Water from Ambion (P/N9915G), change for a constituent in FIG. 11(b) is 10°) when or equivalent) compared to the calibrator. Comparison to the calibrator results in gene expression profiles that are increased, 0199 Methods decreased, or without change from the calibrated Set. 0200 1 Place RNase Inhibitor and MultiScribe Reverse 0212 FIG.11(a) shows relative gene expression (mRNA Transcriptase on ice immediately. All other reagents can be synthesis) in heat-killed Staphylococci (HKS)-stimulated thawed at room temperature and then placed on ice. cells, and the effect of three different compounds (TPCK, 0201 2 Remove RNA samples from -80° C. freezer and UT-77, and “Dex”, or dexamethasone). Compound TPCK thaw at room temperature and then place immediately on caused a 10-fold decrease in relative IFN-Y expression, and ice. 100,000-fold decreases in IL-4 and IL-5 expression. Further, compound UT-77 caused even greater magnitude of 0202 3 Prepare the following cocktail of Reverse Tran increases in relative expression of the gene encoding IL-5, scriptase Reagents for each 100 PLRT reaction (for multiple and more modest increases in IL-1 expression (more than Samples, prepare extra cocktail to allow for pipetting error): 10-fold) and IFN-Y. Such effects can be highly significant in disease etiologies and outcomes, and have predictive value concerning the usefulness as therapeutic agents of these 1 reaction(uL) 11X, e.g. 10 samples(uL) compounds or Similar chemical entities or chemicals that act similarly. HKS cells may be used as an in vitro model of 1OXRT Buffer 1.O.O 11.O.O 25 mM MgCl2 22.O 242.O Gram-positive bacterial infection. dNTPs 2O.O 22O.O 0213 FIG. 11(b) displays analyses of expression of the Random Hexamers 5.0 55.0 RNAse Inhibitor 2.0 22.0 12 genes in lipopolysaccharide-(LPS)-treated cells, an in Reverse Transcriptase 2.5 27.5 Vitro model of Gram-negative bacterial infection. These data Water 18.5 2O3.5 include several striking contrasts to the data in FIG. 11(a). Thus treatment with the therapeutic agent DeX caused a Total: 8O.O 880.0 (80 uL per sample) Striking decrease in expression of the IL-2 gene in LPS treated cells, and a Striking increase in IL-2 expression in 0203 4 Bring each RNA sample to a total volume of 20 HKS-treated cells. Strikingly large differences in gene AiL in a 1.5 mL microcentrifuge tube (for example, for expression in the differently Stimulated cells can be seen for THP-1 RNA, remove 10 ul RNA and dilute to 20 uL with the IL-4 and the IL-5 genes. Expression of the gene for IFN, RNase/DNase free water . . . for whole blood RNA use 20 in contrast, responded Similarly in cells treated by either of uL total RNA) and add 80 ul RT reaction mix from step the Stimuli and any of the therapeutic agents. 5.2.3. Mix by pipetting up and down. 0214. By these criteria, expression of the genes for IL-2, IL-4 and IL-5 were observed to be candidate markers or 0204 5 Incubate sample at room temperature for 10 Surrogate markers in cell model Systems to distinguish minutes. responses of the cells to Gram-positive and Gram-negative 0205 6 Incubate sample at 37° C. for 1 hour. bacterial infection. 0206 7 Incubate sample at 90° C. for 10 minutes. Example 3 0207 8. Quick spin samples in microcentrifuge. 0215. A single therapeutic agent for treating a particular 0208 9 Place sample on ice if doing PCR immediately, condition can be differentiated from a Second therapeutic otherwise store sample at -20° C. for future use. agent that also treats the particular condition by a signature 0209) 10 PCR QC should be run on all RT samples using profile for a given Selected panel of gene loci. 18S and B-actin (see SOP 200-020). 0216 FIG. 12 shows a calibrated profile data set for a panel having 8 constituents that are indicative of a biological Example 2 condition that includes inflammation. The profiles are shown for three different anti-inflammatory agentS-methotrexate, 0210 Different inflammatory stimuli give rise to differ meclofenamate and methylprednisolone. The calibrated pro ent, baseline profile data Sets So that the calibrated Selected file data Sets for each agent as shown represents a Signature profiles for different agents in the same class of anti profile for that agent. This signature profile may serve as a inflammatory result in different Signature profiles. device for establishing quality control for a batch of the 0211 FIGS. 11(a) and 11(b) show different inflammatory agent. Indeed, it is envisaged that compounds or classes of Stimuli give rise to different, baseline profile data Sets that compounds on the market or in development may be char may be used in determining the calibrated Selected profile acterized by a signature profile. The Signature profile may be data Sets for the three anti-inflammatory agents tested, and represented in a graphical format, more particularly as a bar the resulting different Signature profiles. The different pro graph as provided in FIG. 12. For FIG. 12, an ex vivo files reflect the difference in the molecular targets and Sample was tested. A Sample of blood was taken from the mechanisms of action of the three agents derived from a Subject. Aliquots of the Sample were Subjected to Single class of therapeutics, anti-inflammatory agents. FIG. lipopolysaccharide (LPS) ex vivo. After 30 minutes, the 11(a) also illustrate the extraordinary range of detection anti-inflammatory agent as indicated was added to an aliquot (y-axis) from less than 10 fold difference from the calibrated of the sample of blood and after about another 4 hours, the US 2004/0225449 A1 Nov. 11, 2004 expression of the panel of genes (Il-1C, Il-2, Il-8, Il-10, Example 5 Il-12p35, Il-12p40, IL-15, IFN-Gamma and TNF-C) was 0220 Similarities and differences in the effect of a single determined. Although the calibrated profile of methotrexate agent on cell populations differing in their biological con and meclofenamate were similar, the calibrated profile of dition. methylprednisolone was substantially different. Differences may be reflective of the differences of the mechanisms or 0221 Ex-vivo gene expression analysis can be performed target(s) of action of this agent within the general class of by obtaining the blood of a Subject for example by drawing anti-inflammatory compounds. The baseline is the profile the blood into a vacutainer tube with Sodium heparin as an data Set for lipopolysaccharide absent any additional agents. anticoagulant. An anti-inflammatory Such as 3-methyl-pred nisolone at a final concentration of 10 micromolar was Example 4 added to blood in a polypropylene tube, incubated for 30 minutes at 37 C in 5% CO. After 30 minutes a stimuli such 0217. There is relatively low variability with respect to as LPS at 10 ng/mL or heat killed Staphlococcus (HKS) at the profile within a single individual over time when the 1:100 dilution was added to the drug treated whole blood. Incubation continued at 37 C. in 5% CO for 6 hours unless calibrated Selected profile is determined from the measure otherwise indicated. Erythrocytes were lysed in RBC lysis ment of gene expression acroSS many gene loci that have Solution (Ambion) and remaining cells were lysed according been appropriately induced. to the Ambion RNAqueous-Blood module (catalog # 1913). 0218 FIGS. 13(a), 13(b), and 13(c) show graphical rep RNA was eluted in Ambion elution Solution. RNA was resentations of calibrated Selected profile data Sets for two DNAsed treated with 1 unit of DNAse I (Ambion #2222) in different samples of whole blood. Heparinized whole blood 1xDNAse buffer at 37° C. for 30 minutes. In this example, first Strand Synthesis was performed using the Applied from a single normal healthy Volunteer was collected on two Biosystems TaqMan Reverse Transcriptase kit with Multi Separate occasions of more than 2 weeks apart. FIG.13a, for Scribe reverse transcriptase (catalog # NO-0234). Quality sample 991116, and FIG.13b, for sample 991028, reflect the check of RT reactions were performed with Taqman PCR biological condition of the tested cells from the Single donor chemistry using the 18S rRNA pre-developed assay reagents using a selected panel (i.e., the inflammation Selected panel) (PDAR) from Applied Biosystems (part #4310893E). PCR of 24 members, in response to Stimulation with one of three assay of Source Selected Profiles were performed on 6 to 24 different agents. The baseline in this example is derived genes in four replicates on the Applied Biosystems 7700. from untreated cells obtained from the same individual. The PCR assays were performed according to Specifications calibrated profiles are shown for cells exposed for 4 to 6 outlined with the PDAR product. Relative quantitation of hours to lipopolysaccharide (LPS), heat-killed Stapylococci the gene of interest was calibrated against 18S rRNA expres (HKS), and phytohemagglutinin (PHA). FIG. 13c shows a Sion as described in Applied BioSystems product User direct comparison of LPS-stimulated blood sample 99116 Bulletin 2 (1997) and elaborated in Hirayama, et al (Blood with respect to blood sample 991028, i.e., 99.1028 is used as 92, 1998:46-52) using 18S instead of GAPDH. the calibrator or baseline profile data Set. The messenger RNA levels measured on Oct. 28, 1999 were used to 0222 Relative quantitation of the mRNA was measured compare the levels of messenger RNA measured on Nov. 16, by the difference in threshold cycles between 18S and the 1999. A perfect identity of RNA levels would be represented gene of interest. This delta CT was then compared to the by a flat line at unity. These data show that for baseline gene normalizing condition, either Subject before treatment, or expression, there can be as much as an 8 fold difference Stimuli without drug in an ex-Vivo assay to measure "fold (c-jun) in messenger RNA levels. However, for most of the induction” represented in the bar graphs (FIG. 14). For genes measured, when there is no known Substantial physi example, in the above graph, IFN- levels are /SO leSS on ological change in the Subject, the levels of messenger RNA day 3 than before treatment. measured on one day are Similar to those measured on a different day. Changes in gene expression, whether mRNA Example 6 or protein, in excess of 10-20% may be reflective of bio 0223) In Vivo and Ex vivo samples provide comparable logical changes in the Subject even though traditional clini Signature profiles. cal measurements may not identify Such changes. FIG. 13(d) is similar to FIG. 13(c) except that the cells were not 0224 FIG. 15 shows the calibrated profile data set for Stimulated with LPS. two subjects (Subject 1 and Subject 2) who have been treated over a three day period with a Standard dose of the 0219 FIGS. 13(a) through 13(d) document the relatively corticosteroids, dexamethasone. Blood from each Subjects low variability with respect to the profile within a single was obtained 72 hours later and a quantitative measure of the individual over time in Similar physiological conditions amount of RNA corresponding to the panel constituents was when the calibrated selected profile is determined from the determined. Although, the calibrated profile data Set for each measurement of gene expression acroSS many gene loci that Subject was similar for most gene loci, Some notable differ have been appropriately induced. The figures illustrate (1) ences were also detected, for example for Il-2, Il-10, Il-6 and the class-specific effects (generally inflammatory as deter GM-CSF. A calibrated profile data set is also shown for mined by the effect on pro-inflammatory gene loci, e.g. comparison for an eX Vivo Sample of blood from Sample 1 TNF-alpha, IL-1 alpha and IL-1 beta), (2) the agent-specific prior to treatment with corticosteroid where the ex vivo effects quantitative differences between each of the agents at Sample is Subjected to an equivalent amount of corticoster the same gene loci (e.g., IL-2) and (3) reproducible and oid in vitro as calculated to be the plasma level in the therefore predictable effects on the subject population, TK subject. The similarity in the calibrated profile data set for ex (FIG. 13c). Vivo Samples when compared to in Vivo Samples provides US 2004/0225449 A1 Nov. 11, 2004 22
Support for an in vitro assay that will predict the in vivo herbal while providing information concerning its properties action of the compound. We have observed a similar com and its efficacy for a Single Subject or for an average parable effect between in vivo and ex vivo samples infected population of Subjects. with an infectious agent, more particularly bacterial or viral agents. We have concluded therefore that the ex vivo Example 9 Samples provide an effective method of determining the effect of a Single compound or multiple compounds on a 0230. A quality control assay for Echinacea brands using patient, where the multiple compounds may be either used calibrated profile data Sets. in combination, in parallel or Sequentially to optimize the 0231 FIG. 24 shows a graphic representation of the Selection of an agent for a biological condition for the calibrated profile data sets for four different commercial Subject. brands of Echinacea. Brands using an Inflammation Selected Panel. As expected, SPM007 and SPM003 gave the signa Example 7 ture, calibrated profiles Similar to authentic Echinacea. Samples SPMO10 and SPM 016, although labeled and sold 0225 Demonstration of reproducibility of an in vitro as Echinacea when tested using the System described in response with an approved anti-inflammatory on 5 different FIG. 14, resulted in signature calibrated profiles that were donor Subjects. Substantially Similar to the profile obtained with lipopolysac 0226 Comparison and analysis of the FIGS. 18a through charide alone. Echinacea samples SPMO10 and SPMO16 18e demonstrate the consistency of effect of the stimulus and were found to have elevated, highly biologically active in vitro treatment with an approved anti-inflammatory on 5 levels of endotoxin while the LPS levels in SP007 and different donors (each figure representing a unique donor). SP003 were undetectable. A stored signature profile for The use of a known and tested Stimulus results in a highly active Echinacea obtained from a Selected panel designed to reproducible gene response in vitro that may be correlated test efficacy and mode of action, e.g., the inflammation with a predictable in Vivo response. panel, permits evaluation of new batches of Echinacea, differentiation of existing or new brands of Echinacea, guide 0227 FIGS. 18a-18e provide the results of analysis of 5 the isolation and development of new compounds with donors from which a blood sample has been taken. The different or Similar activities from a complex compound like blood Samples were exposed to a therapeutic agent at Echinacea or may be used in the development of quality various concentrations ranging from 0.1 uM to 5 uM, more asSurance in the production, analysis and Sale of new or particularly 0.1 uM, 0.3 uM, 1 uM, 3 uM and 5 uM, for a 4 previously marketed compounds. In the example cited, two hour period. Different concentrations of the drug resulted in of the brands of Echinacea SPO10 and SPO16.result in a calibrated profile data Set for an inflammation panel at each calibrated profiles that are characteristic of authentic Echina concentration that was qualitatively different from the next. CC2. FIG. 18a corresponds to donor 1, FIG. 18b corresponds to donor 2, FIG. 18c corresponds to donor 3, FIG. 18d Example 10 corresponds to donor 4, and FIG. 18e corresponds to donor 5. Each individual varied from the other and also provided 0232 Comparison of three herbal preparations using an a variable profile for a different concentration. This set of indicator cell line. figures illustrates the high level of information obtainable by 0233 FIGS. 25(a) through 25(c) provide calibrated pro calibrated profile data Sets. file data Sets for three herbal preparations with respect to an indicator cell line (THP-1) rather than a blood sample from Example 8 a subject. In FIG. 25(a), the baseline is the profile data set 0228. A calibrated profile data set may provide a signa for THP-1 cells absent the herbal while the histograms ture profile for a complex mixture of compounds. represent the calibrated profile data Sets for the same herbal from three different manufacturing Sources of the same herb 0229 FIG. 21 illustrates the effect of three different at 250 ug/ml. Gene expression results are shown on a log anti-inflammatory herbs on a Selected panel of constituents Scale. Similar to the observation in FIG. 14, these demon including constituents of an Inflammation Selected Panel strate that similarly labeled compounds obtained from dif (TNF-C, Il-1b, ICAM, Il-8, Il-10, Il-12p40, ICE, cox-2, ferent Sources have demonstrable and quantifiable differ cox-1 and mmp-3) a cell growth and differentiation Selected ences in calibrated profiles using a Specific panel, e.g., the panel (c-fos, c-jun and STAT3), a toxicity Selected panel inflammation Selected panel designed to obtain information (SOD-1, TACE, GR, HSP70, GST, c-fos, c-jun, INOS) and about the expression of gene products related to inflamma a liver metabolism Selected panel (INOS, cyp-a and u-pa). tion and infection. This Suggests that the compounds likely The cells assayed in FIG. 21 are aliquots of blood from a have different efficacies when used for Specific purposes. Subject that are exposed eX Vivo to lipopolysaccharide and to Echinacea (SPM9910214) Arnica (SPM991.0076) and Sibe 0234 FIG. 25(b) provides a comparison of the calibrated rian Ginseng (SPM9910074), each of the nutraceuticals profile of a Single herb at three concentrations using the being applied to the blood Sample at the same concentration indicator cell line of THP-1. The baseline profile data set is of 200 ug/mL. The baseline is cell sample with lipopolysac untreated THP-1 cells. Analysis of the data Suggests a charide in the absence of a nutraceutical. Each nutraceutical concentration-dependent response in the indicator cell lines (formed from a complex mixture) has a characteristic Sig which, although demonstrated here, may be indicative of a nature profile just as did the Single compound pharmaceu Similar response in Subjects. tical anti-inflammatory agents. The Signature profile may be 0235 FIG. 25(c) provides a comparison of four com provided in a graphic form that can be use to identify a mercial Echinacea brands used at the Same concentration US 2004/0225449 A1 Nov. 11, 2004 23 and tested against a panel of constituents using a THP-1 cell chosen for its ability to discriminate as to the effect of line as an indicator cell population. Differential expression, anti-inflammatory agents. For each constituent, the post as revealed, for example, by inspection or calculation of administration concentration is shown as a ratio in relation differences in the calibrated profiles, allows direct compari to its pre-administration concentration; hence the baseline of Sons of complex compounds to be made. For example, 1 is indicative of the same concentration of the constituent analysis of the differences in the calibrated profiles may be before and after administration, and the presence of a bar used to guide compound isolation and development, product below the baseline indicates a post-administration drop in differentiation in the marketplace, or used by the consumer concentration of the constituent. FIGS. 26(a) and 26(c) show or health professional to guide the individualized choice of that the response of Subjects 1 and 2 to the administration of a single compound from a class of Similar compounds that the Steroid are qualitatively and quantitatively similar-in may be Suited for a particular biological condition. deed, Strikingly similar. Moreover, and in contrast, the responses of controls 1 and 2 to the placebo are markedly Example 11 distinct from the responses of Subjects 1 and 2. 0236 Set up of a 24-gene Human Selected Panel for Inflammation. Example 13 0242 Calibrated profile data sets, using a subset of the 0237) Materials Inflammation Selected Panel, provide a comparison of the effects of administration of methylprednisone and Ibupro fen. In this example, FIGS. 27(a) and 27(c) are identical to 1 20X Primer/Probe Mix for each gene of interest. FIGS. 26(a) and 26(c), and show the responses of subjects 2 20X Primer/Probe Mix for 18S endogenous control. 1 and 2 respectively to the administration of methylpred 3 2X Taqman Universal PCR Master Mix 4 cDNA transcribed from RNA extracted from cells nisone. FIGS. 27(b) and 27(d) show the responses of the 5 Applied Biosystems 96-Well Optical Reaction Plates Same Subjects, namely Subjects 1 and 2 respectively, to the 6 Applied Biosystems Optical Caps, or optical-clear film administration of high-dose Ibuprofen (800 mg administered 7 AB Prismal 7700 Sequence Detector three times per day over a three-day period). (FIGS. 27(b) and 27(d) use the same conventions as FIGS. 27(a) and 27(c) in showing post-administration concentration of con 0238 1 Make stocks of each Primer/Probe mix contain Stituents relative to pre-administration concentration.) It can ing the Primer/Probe for the gene of interest, Primer/Probe be seen from these figures that the responses of the Subjects for 18S endogenous control, and 2x PCR Master Mix as to Ibuprofen are qualitatively and quantitatively similar follows. Make Sufficient excess to allow for pipetting error again, Strikingly similar. Moreover, the responses of the e.g. approximately 10% excess. The following example Subjects to Ibuprofen are distinct from the responses of the illustrates a typical Set up for one gene with quadruplicate Subjects to methylprednisone. In fact, the distinct pattern of Samples testing two conditions (2 plates). response to Ibuprofen correlates with other information known about Ibuprofen. For example, IL-1-?3 is known to be of importance in responding to joint destruction, and Ibu profen here is shown as not raising the level (and in the case 1X(1 well) 9X (2 plates worth) of Subject 2, lowering the level) of IL-1-?3 expression. And 2X Master Mix 12.50 112.50 Ibuprofen is known to be not very effective in treating joint 2OX 18S Primer/Probe Mix 1.25 11.25 destruction. Similarly, IL-10 activity is associated with 2OX Gene of interest Primer/Probe Mix 1.25 11.25 anti-inflammatory activity and is useful in addressing bowel Total 15.OO 135.OO inflammatory disease, Ibuprofen here is shown as in fact depressing the level of IL-10 expression. These phenomena are consistent with the fact that Ibuprofen is known to be 0239). In Examples 12 through 19 below, procedures ineffective in addressing bowel inflammatory disease. analogous to those of Examples 1 and 11 were followed to determine relative mRNA expression. Example 14 Example 12 0243 Calibrated profile data sets, using a subset of the Inflammation Selected Panel, identify chronic obstructive 0240 Calibrated profile data sets, using a subset of the pulmonary disease (COPD) patients. Inflammation Selected Panel, show the effect of administra 0244. The graphs of these each of FIGS. 28(a) through tion of a Steroid. 28(d) show the relative concentration of each constituent of 0241. In FIGS. 26(a) and 26(c), Subjects 1 and 2 respec a subset of the Inflammation Selected Panel, the Subset being tively have been Subjected to a course of administration of chosen for its ability to discriminate as to the presence of methylprednisone twice a day for three consecutive days. In COPD. For each constituent, the concentration is shown as FIGS. 26(b) and 26(d), two other individuals, identified as a ratio in relation to a concentration that is normative of the control 1 and control 2 respectively, were administered a concentrations of the constituent in a population of healthy placebo over a corresponding period. In each case, the blood Subjects. Thus for any constituent, a baseline level of 1 Samples of the individuals were taken prior to the course of corresponds to a normal concentration. FIGS. 28(a) and administration and immediately following the course of 28(c) show the relative concentrations of constituents in administration. The graphs of these each of these figures COPD patients 1 and 2 respectively, while FIGS. 28(b) and show the relative concentration of each constituent of a 28(d) show the relative concentrations of constituents in two Subset of the Inflammation Selected Panel, the subset being healthy individuals identified as control 1 and control 2 US 2004/0225449 A1 Nov. 11, 2004 24 respectively. Indeed, FIGS. 28(b) and 28(d) show levels of because its metabolism in the rat and human liver is well constituents in controls 1 and 2 as being close to population described. AS expected, clofibrate induces gene expression normals, whereas FIGS. 28(a) and 28(c) show levels of at the cyp 1A1 locus, but the agent also induces expression constituents in COPD patients 1 and 2 as being dramatically at a number of other metabolic loci in the Selected panel as different from normal levels. measured in this cohort of in-bred Spraque-Dawley rats. The ratio of the concentration of each constituent for the clofi Example 15 brate treated rats is measured with reference to a control (baseline) which is a set of rats treated only with the carrier 0245 Evaluations, of the effects of drug exposure per compound. The resultant selected profile is provided in FIG. formed in Vitro correspond closely with evaluations per 31. formed in Vivo, employing in each case calibrated profile data Sets, using a Subset of the Inflammation Selected Panel. Example 18 0246 FIGS. 29(a) and 29(b) present graphs showing 0251 The ability of the rat metabolism selected panel to response to the administration of methylprednisone. The differentiate drug responses response shows the relative concentration of each constitu ent of a Subset of the Inflammation Selected Panel, the 0252 Male rats were treated with 400 mg/kg/day of Subset being chosen for its ability to discriminate as to the clofibrate or benzoapyrene administered by mouth and the effect of anti-inflammatory agents. For each constituent levels of gene expression were measured in liver tissue. The illustrated, the darker bar (on the right) shows the in vivo response to clofibrate and benzoapyrene was determined in response of a Subject to a course of administration of Spracque-Dawley rats using a rat metabolism panel. The methylprednisone twice a day for three consecutive days. In results are shown in FIG. 32. Each drug gives a character the in Vivo cases, a blood Sample of the Subject was taken istic and distinct pattern of gene induction acroSS the prior to the course of administration and immediately fol Selected panel. AS expected from the prior art, benzoa lowing the course of administration, the constituents were pyrene Specifically induces gene expression at the loci for measured, and the responses are shown in FIGS. 29(a) and cyp 4A1 and HD. The control (baseline) is a set of rats 29(b). The post-administration concentration of each con treated only with the carrier control. Stituent is shown as a ratio in relation to its pre-administra tion concentration; hence the baseline of 1 is indicative of Example 19 the same concentration post-administration as pre-adminis 0253) The effect of administration of a stimulant is mea tration. The procedures of FIGS. 29(a) and 29(b) were Sured by a skin-epithelial/vascular/inflammation Selected conducted on two different occasions a year apart. At the panel. Same time that each in Vivo procedure was conducted, methylprednisone was also administered in Vitro to a Sample 0254 FIG. 33 illustrates the response of a subject to the of the blood of the same subject. For each constituent administration of a stimulant (TNF-alpha, 10 ng/ml), as illustrated in each of FIGS. 29(a) and 29(b), the lighter bar measured by a skin-epithelial, Vascular/inflammation (on the left) shows the in vitro response of the sample to the Selected panel. In this example, the Selected panel is created administration of the drug. Again, the post-administration from constituents that are also found in other panels which concentration of each constituent is shown as a ratio in have been here Selected for purposes of best establishing an relation to its pre-administration concentration. effect resulting from the Stimulant. 0247 What is remarkable about the results shown in FIGS. 29(a) and 29(b) is that in each procedure, the in vitro Example 20 response is Strikingly similar to the in Vivo response, in most 0255 Use of a human liver selected panel for determin cases even where the results in vivo differed over time. This ing the metabolic properties of cryopreserved human hepa result shows the value of in vitro modeling for the evaluation tocytes. of the effect of the administration of agents using Selected Panels. 0256 Time/dose response experiments utilizing com pounds with well-described toxicities and mechanisms of Example 16 action are an early Step in the biological validation of the selected panel. FIG. 34 shows the gene expression profile 0248. The effect of different agents is evaluated using a resulting from a timed Study at constant dose of benzo(a)py Subset of the Selected Prostate Panel. rene when administered to cryopreserved human hepato 0249. In FIG. 30 is shown the response of five different cytes. cell lines to the administration of various agents, using a Subset of the Selected Prostate Panel (listed in Table 5). This Example 21 figure also shows broad functions of constituents of the 0257 Response of human umbilical vein endothelial panel. cells to TNF-C. Example 17 0258 FIG.35 illustrates how endothelial cells respond to the inflammatory TNFC. by the induction of expression of a 0250) The use of a rat liver metabolism selected panel to number of gene loci, notably the adhesion molecules measure the effect of a pharmaceutical agent, clofibrate, ELAM, ICAM, and VCAM. The cells were exposed for 24 Male rats were treated with 400 mg/kg/day of clofibrate hours. Both 5 ng/ml and 10 ng/ml are high doses of the administered by mouth and the levels of gene expression immunostimulant TNFC. and, as expected, no clear concen were measured in liver tissue. Clofibrate is used here tration response is observed at this dose level. US 2004/0225449 A1 Nov. 11, 2004 25
Example 22 0267. Different inflammatory stimuli give rise to different 0259. This example is one illustration of the wide profile data Sets that may be used in determining the Selected embodiments of the Selected panels. In this example the profile data Sets for whichever anti-inflammatory agents are effect of a compound in solution (NAC) is compared directly tested, and the resulting different Signature profiles. The to the effect of an environmental stimulus (UVB) and the different profiles reflect the difference in the molecular combined effect is read out as differential gene expression. targets and mechanisms of action of the different agents FIG. 19 illustrates an similar effect of NAC only in blood derived from a single class of therapeutics, i.e., anti-inflam obtained SmokerS and non-SmokerS. Dose and time experi matory agents. An extraordinary range of detection, from less than 10-fold difference from the profile with respect to ments were conducted prior to this illustrated experiment. some constituents to a change of 10" (10E13) in gene Example 23 expression of one constituent is not unprecedented, when 0260 Gene expression profiles provide information on compared to the baseline profile data set (no Stimulus). the effect of an environmental Stimulus on cells. Comparison to the baseline results in gene expression pro 0261) This example is one illustration of the wide files that are increased, decreased, or without change from embodiments of the Selected panels. In this example (see the baseline Set. FIG. 36) the effect of a compound in solution (N-acetyl 0268 Such large and small effects can be highly signifi cysteine) is compared directly to the effect of an environ cant in disease etiologies and outcomes, and have predictive mental stimulus (UVB) and the combined effect is read out value concerning the usefulneSS as therapeutic agents of as differential gene expression. FIG. 19 illustrates an similar Specific compounds, or those with Similar chemical entities, effect of NAC only in blood obtained smokers and non or those chemicals that act Similarly. SmokerS. Dose and time experiments were conducted prior to this illustrated experiment. Example 26 Example 24 0269 Gene expression profiles provide information on 0262 Demonstration of reproducibility of an in vitro the effect of an environmental Stimulus on cells. response with an approved anti-inflammatory on different 0270. As an illustration of the wide embodiments of donor Subjects. using a Selected panels, the effect of a compound in Solution 0263. The consistency of effect of the stimulus and in is compared directly to the effect of an environmental Vitro treatment with an approved anti-inflammatory on dif stimulus (UVB) and the combined effect is read out as ferent donors is performed by taking blood Samples, as differential gene expression. A similar effect of a compound described above in Example 1, and preparing aliquots for may be observed in blood obtained from smokers and baseline, non-Stimulus, and Stimulus with Sufficient Volume non-SmokerS. Dose and time experiments are conducted for at least three time points. Typical Stimuli include ibu prior to this type of experiment, to optimize concentrations profen, acetaminophen, aspirin, prednilisone, prednisone or of compounds to be investigated, or to define useful expo dexamethasone and may be used individually (typically) or Sure conditions for the environmental Stimulus. Such a in combination. The blood Samples are exposed to an method allows profile data Sets to be obtained from Samples anti-inflammatory therapeutic agent at various concentra from individuals, before exposure and/or treatment, after tions ranging from 0.1 uM to 5 uM, more particularly 0.1 exposure and/or treatment, and provides a description of the LiM, 0.3 uM, 1 uM, 3 uM and 5 uM, for a 4 hour period. biological condition of the Sample at the time and circum Different concentrations of the drug will provide a different stance it is obtained, which in turn provides a description of profile data Set for an inflammation panel, comprised of two the biological condition of the individual donor, both before or more genes from Tables 1-8, at each concentration that is and after treatment and/or exposure. qualitatively different from the next. At defined times, cells are collected by centrifugation, the plasma removed and TABLE 1. RNA extracted by various Standard means, amplified and Inflammation Selected Panel quantitated, as described in Example 1 above. From the IL-1C. Interleukin-1 alpha quantitative measurements of amplified RNA, profile data IL-1B Interleukin-1 Beta Sets for the Selected panel are derived for each aliquot. IL-2 Interleukin-2 IL-4 Interleukin-4 0264. Each resulting profile data set is descriptive of the IL-6 Interleukin-6 biological condition of the Sample at the time and circum IL-7 Interleukin-7 stances at which the Sample is taken, and therefore descrip IL-8 Interleukin-8 IL-10 Interleukin-10 tive of the biological condition of the donor from which the IL-12p40 Interleukin-12p40 Sample is taken. IL-15 Interleukin-15 IL-18 Interleukin-18 0265. Each sample from the individual donors varies GM-CSF Granulocyte colony stimulating factor from the other and also provides a variable profile for a IFN-y Interferon gamma different concentration. Such a technique allows a high level TGF-C. Tumor growth factor alpha of information obtainable by profile data sets. TNF-C. Tumor necrosis factor alpha TNF-B Tumor necrosis factor beta Example 25 Cox 2 Cyclooxygenase/prostaglandin-endoperoxide synthase 2 0266 Different inflammatory stimuli give rise to different ICE Interleukin-1 converting enzyme profile data sets so that the selected profiles for different c-jun MKK7, MAP2K7 agents in the same class of anti-inflammatory result in mmps Matrix metalloproteinase different Signature profiles. US 2004/0225449 A1 Nov. 11, 2004 26
0272 TABLE 1-continued TABLE 3 Inflammation Selected Panel Human Liver Metabolism and Toxicity Selected Panel (See FIG. 34) UPA Urokinase plasminogen activator HSP70 Heat Shock Protein 70 kDa CYP1A2 Polycyclic aromatic hydrocarbon (PAH) CRE cAMP Response Element metabolism; induced by smoking ICAM Intercellular Adhesion Molecule Catalyzes formation of toxic APAP metabolite Catalyzes oxidation in some pharmaceuticals, procarcinogens, and smoke constituents; upregulated in vitro 0271) after exposure to barbiturates or dex. Converts many small organic TABLE 2 Compounds (i.e. EtOH, APAP, CC14) into reactive intermediates. Induced in Cell Growth and Differentiation Selected Panel alcoholics and in fatty liver phenotype. CYP2D6 Broad catalytic activities for over 30 BIRC5 (Survivin) Apoptosis Inhibitor therapeutic drugs. NFKB1 NF-kappaB Metabolism for a wide variety of drug types CDKN2A (P16) Cell cycle inhibitor UDP glycosyltransferase 2B7 TP53 (P53) Tumor suppressor UDP glycosyltransferase 2B15 TNF-C. Tumor Necrosis Factor alpha Microsomal epoxide hydrolase TERT Telomerase Catalytic Subunit Multiple tissue-specific splicing variants BCL2 Represses Apoptosis GSTA1 Glutathione S-transferase alpha 1 BAX Promotes Apoptosis GSTA2 Glutathione S-transferase alpha 2 CASP1 (ICE) Interleukin Converting Enzyme UCP-2 Mitochondrial uncoupling protein 2 GADD45A Growth arrest protein Local inflammation TNFRSF11A (RANK) Receptor activator of NFkB Endothelial activation PDCD8 (AIF) Apoptosis Inducing Factor Released by PBMCs, Kupffer cells and Apaf-1 Apoptotic protease activating factor 1 activated tissue macrophages in the liver DFFB (DFF40) Caspase activated DNAse TGF-B Transforming growth factor beta 1 BAIAP3 (IAP1, BIRC3) Inhibitor of apoptosis protein NOS Inducible nitric Oxide synthase; 1 (BAI-associated protein 3) SCF Stem cell factor, released by activated BIRC2 (IAP2) Inhibitor of apoptosis protein 2 hepatic stellate cells (HSC); recruits Bik BCL2 interacting killer mast cells (MC) to the liver BCL2L1 (BCL-X) BCL2-Like 1 IFN-y Activation of macrophages DAD1 Defender against cell death 1 Galectin-3 f-galactoside-binding lectin associated MADD MAP Kinase activating death domain with cell growth, tumor transformation, MAP3K14 Mitogen-activated protein kinase 3, 14 and metastasis. PTEN Protein tyrosine phosphatase FAP Fibroblast activation protein; membrane k-alpha-1 Alpha tubulin (housekeeping, high abundance) protease expressed at sites of tissue TOSO Anti-fas induced apoptosis remodeling. cdk2 Cyclin dependent kinase 2 Procollagen C-proteinase Extracellular matrix protein cdk4 Cyclin dependent kinase 4 (aka BMP1) (bone morphogenetic protein 1) CASP 3 Apoptosis-related cysteine protease, 3 required for cartilage formation CASP 9 Apoptosis-related cysteine protease, 9 Collagen I Extracellular matrix (ECM) component RAD52 DNA ds break repair Collagen III ECM component XRCC5 (KuSO) X-ray repair complementing defective repair Collagen IV ECM component in Chinese hamster cells 5 Laminin ECM component PNKP Polynucleotide kinase phosphatase MRE11A Meiotic recombination 11 homolog A Fibronectin ECM component CCND1 (cyclin D1) PRAD1: parathyroid adenomatosis 1 CCND3 (cyclin D3) Cyclin D3 CCNE1 (cyclin E) Cyclin E CCNA2 (cyclin A) Cyclin A 0273) CCNB1 (cyclin B) Cyclin B CDKN2B (p15) Cyclin-dependent kinase inhibitor 2B TABLE 4 (p15, inhibits CDK4) CDKN1A (p21) Cyclin-dependent kinase inhibitor 1A Skin Response Selected Panel (p21, Cip1) RB1 Retinoblastoma CRABP2 Cellular retinoic acid-binding protein 2 BID BH3 interacting domain death agonist KRT14 Keratin 14 BAK1 BCL2-antagonist/killer 1 KRTS Keratin 5 BAD BCL2-antagonist of cell death KRT16 Keratin 16 SMAC Second mitochondria-derived activator of FGF7 Fibroblast growth factor (KGF) Keratinocyte growth factor caspase FN1 Fibronectin 1 VDAC1 Voltage-dependent anion channel 1 IVL Involucrin CHEK1 Checkpoint, S. pombe, homolog of, 1 COL7A1 Type VII collagen, alpha 1 ABL1. V-abl Abelson murine leukemia viral oncogene CTGF Connective tissue growth factor homolog 1 IL-1C. Interleukin-1C. TNFRSF12 Tumor necrosis factor receptor superfamily, IL-8 Interleukin 8 member 12 GRO1 Melanoma growth stimulatory activity (MGSA) TRADD TNFRSF1A-associated via death domain PTGS2 Prostaglandin-endoperoxide synthase 2 (COX2) FADD Fas (TNFRSF6) - associated via death domain Cyclooxygenase 2 TRAF1 TNFR associated factor 1 TNF-C. Tumor necrosis factor alpha TRAF2 TNFR associated factor 2 TGF-B1 Transforming growth factor beta 1 PI3 Proteinase inhibitor 3 US 2004/0225449 A1 Nov. 11, 2004 27
TABLE 4-continued TABLE 5-continued Skin Response Selected Panel Prostate Selected Panel (See FIG. 30) MRP1 Multiple Drug Resistance Protein 1 (SKALP) Skin-derived antileukoproteinase HSP-70 Heat shock protein BSG Basignin (EMMPRIN) Extracellular MMP inducer TNF-C. Tumor Necrosis Factor MMP1 Matrix metalloproteinase 1 (interstitial collagenase) MMP2 Matrix metalloproteinase 2 (72 kD gelatinase) MMP3 Matrix Metalloproteinase 3 (Stromelysin. 1) TIMP1 Tissue inhibitor of matrix metalloproteinase 0275 HMOX1 Heme Oxygenase1 GADD45A Growth arrest and DNA-damage-inducible alpha TABLE 6 PCNA Proliferating cell nuclear antigen DUSP1 Dual specificity phosphatase(CL100) Vascular Selected Panel MAPK8 Mitogen activated protein kinase 8 TP53 Tumor protein p53 (p53) VEGF Vascular Endothelial Growth Factor BC2 B-cell CLL/lymphoma 2 NF kappa B Nuclear Factor kappa B TEK/TIE2 Tyrosine kinase, endothelial Bax Bcl2-associated X protein ERK2 MAPK1: mitogen-activated protein kinase 1 JUN c-jun SELE selectin E (endothelial adhesion molecule 1) FOS c-fos Flt-1 fms-related tyrosine kinase 1 (vascular endothelial NR12 Nuclear receptor subfamily 1, group I, member 2 (PAR2) growth factor/vascular permeability factor receptor) Protease activated receptor 2 PTX3 pentaxin-related gene, rapidly induced by IL-1 beta S100A7 S100 calcium-binding protein A7 (PSOR1) psoriasin 1 HMOX-1 HMOX1 = heme oxygenase (decycling) 1 HIF-1 Hypoxia-inducible factor 1, alpha subunit TNSF6 Tumor necrosis factor (ligand) superfamily, member 6 GRD1 Glutathione Reductase 1 (FASL) Fas ligand NOS Inducible nitric oxide synthase ET1 Endothelin 1 ECE-1 endothelin converting enzyme 1 PLAT plasminogen activator, tissue 0274) ADAMTS1 a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 PTGIS prostaglandin 12 TABLE 5 (prostacyclin) synthase COX-2 prostaglandin-endoperoxide synthase 2 (prostaglandin Prostate Selected Panel (See FIG. 30) G/H synthase and cyclooxygenase) PSA Prostate Specific Antigen VCAM vascular cell adhesion molecule 1 DD3 Prostate cancer antigen 3 IL-8 Interleukin 8 Survivin Apoptosis Inhibitor 4 Il-1B Interleukin-1beta PSMA Prostate Specific Membrane Antigen IGFBP3 Insulin-like growth factor binding protein 3 Folate Hydrolase 1 GA1 gap junction protein, alpha 1, 43 kD TERT Telomerase Reverse Transcriptase ICAM- 1 intercellular adhesion molecule 1 Telomerase Catalytic Subunit KLK2 Human Kallikrein 2 PDEF Prostate-Derived Ets Factor PSCA Prostate Stem Cell Antigen 0276) POV1 Prostate Cancer Overexpressed Gene 1 PART1 Prostate Androgen-Regulated Transcript 1 TABLE 7 MYC c-myc NRP1 Neurophilin 1 Rat Liver Metabolism and Toxicity Panel (See FIGS. 31 and 32) KAI1 Human Metastasis Suppressor Gene LGALS8 Galectin 8 1 ALDH p16 Cyclin-Dependent Kinase 2A 2 aldose reductase GSTT1 Glutathione-S-Transferase theta 1 3 ARG PA1 Plasminogen Activator Inhibitor 1 4 CYP1A1 bcl-2 B-cell CLL/Lymphoma 2 5 CYP1B1 STAT3 Transcriptional activator 6 CYP2A2 IL-6 Interleukin 6 7 CYP2B2 u-pa Urokinase-Type Plasminogen Activator 8 CYP2C11 KRT-5 Keratin 5 9 CYP2D2 TGF-B Transforming Growth Factor Beta 10 CYP2E1 IL-8 Interleukin 8 11 CYP2A1 VEGF Vascular Endothelia Growth Factor 12 CYP3A1 ACPP Acid phosphatase, prostate 13 CYP1A2 KRT-19 Keratin 19 14 CYP4A1 CK-8 Cytokeratin 8 15 CYP4A3 Maspin Protease Inhibitor 5 16 CYP4F1 HMG-I/Y Non-histone chromosomal protein 17 cytochrome P450 oxidoreductase IGFR1 Insulin Growth Factor Receptor 1 18 epoxide hydrolase HUPAP Human Prostate-Associated Protease 19 HD P53 Tumor suppressor 2O MAO-B COX-2 Cyclooxygenase 2 21 quinone reductase E-CAD e-cadherin 22 alpha-1-AGP N-CAD n-cadherin 23 PPAR-alpha CTNNA1 C-1 catenin 24 GGT PCANAP7 Prostate cancer associated gene 7 US 2004/0225449 A1 Nov. 11, 2004
0277
TABLE 8 Master List for Gene Expression Panels Symbol Alias(es) Name Classification ABCC1 MRP, MRP1, GS- ATP-Binding Cassette, Liver Health Indicator X, ABCC, ABC29 Sub-Family C, Member 1 ABL1. ABL, JTK7, c- V-abl Abelson Murine Oncogene/Enzyme: ABL, p150 Leukemia Viral Oncogene kinase Homolog 1 ACPP PAP Acid Phosphatase, Prostate Enzyme: phosphatase ACTB b-actin, Actin, Actin, Beta Cell Structure cytoplasmic 1, beta cytoskeletal actin ADAM17 CSVP, TACE, A Disintegrin and Metalloproteinase TNF-a converting Metalloproteinase Domain enzyme 17 ADAMTS1 METH1, C3-C5, A Disintegrin-Like and Metalloproteinase KIAA1346 Metalloproteinase (Reprolysin Type) with Thrombospondin Type 1 Motif, 1 AHR Ah receptor, AhR Aryl Hydrocarbon Receptor Receptor/Transcription Factor ALB PROO883 Albumin Liver Health Indicator ALOX5 5-(Glipoxygenase, Arachidonate 5 Enzyme: lypoxygenase 5-GOLO, 5-LO, LipOxygenase LOGS AMACR RACE Alpha-Methylacyl-CoA Proteinase: thiol Racemase ANXA11 CAP-50, ANX11, Annexin A11 Phospholipid Binding Annexin XI, 56 kDa Protein autoantigen APAF1 CED4, KIAAO413 Apoptotic Protease Proteinase Activator Activating Factor 1 APOE Apo-E Apollipoprotein E Ligand ARG2 E > C - 3.5.3.1 Arginase Enzyme B7: B7H2, B7RP1, B7 Protein Cell Signaling and GL50, *Not a Activation HUGO gene symbol BAD BCL2L8, BBC2, BCL2 Agonist of Cell Membrane Protein? BBC6, Death Apoptosis BCLX/BCL2 binding protein BAK1 BAK, CDN1, BCL2-Antagonist/Killer 1 Membrane Protein? BCL2L7, Cell death Apoptosis inhibitor 1 BAX Apoptosis regulator BCL2-Associated X Protein Membrane Protein? Bax Apoptosis BCL2 Apoptosis regulator B-Cell CLL/Lymphoma 2 Membrane Protein? Bcl-2 Apoptosis BCL2L1 BCL-XLIS, BCL2-Like 1 (Long Form) Membrane Protein? Apoptosis
BCL3 BLC4, B-cell B-Cell CLL/Lymphoma 3 Transcriptional Regulator leukemia/lymphoma 3 BID None BH3-Interacting Death Ligand/Apoptosis Domain Agonist BK BIP1, BP4, NBK, BCL2-Interacting Killer Apoptosis BBC1 BIRC2 API1, CIAP1, C- Baculoviral IAP Repeat Apoptosis Inhibitor IAP, IAP1, MIHB, Containing 2 MHC BRC3 API2, C-IAP1, Baculoviral IAP Repeat Apoptosis Inhibitor IAP2, MIHB; Containing 3 MIHC, cIAP2 BRCS Survivin, API4, Baculoviral IAP Repeat Apoptosis Inhibitor EPR-1, IAP4, SVV Containing 5 BPI bactericidal/permeability-increasing protein BSG EMMPRIN, 5F7, Basignin (OK Blood Group) Cell Signaling and CD147, OK, M6, Activation/Membrane TCSF Protein US 2004/0225449 A1 Nov. 11, 2004 29
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification C1OA1, Serum Complement Component 1, Q Complement C1O Subcomponent, Alpha Component Polypeptide None Complement Component 1, Q Complement Subcomponent, Beta Component Polypeptide CALCA Calcitonin?calcitonin-related Cell-signaling and polypeptide, alpha activation CASP1 ICE, IL-1BC, Caspase 1 Proteinase/Apoptosis IL1BC, IL1BCE, IL1B-convertase, P45 CASP3 Yama, Apopain, Caspase 3 Proteinase/Apoptosis CPP32, CPP32B, SCA-1 CASP9 APAF3, MCH6, Caspase 9 Proteinase/Apoptosis ICE-LAP6 CAT EC 1.11.16 Catalase Enzyme: redox CCL2 SCYA2, MCP1, Chemokine (C-C Motif) Chemokine HC11, MCAF, Ligand 2 MGC9434, SMC CF CCL3 SCYA3, LD78 Chemokine (C-C Motif) Chemokine Alpha, MIP1A, Ligand 3 SIS-beta, GOS19-1 CCL5 SCYA5, Chemokine (C-C Motif) Chemokine D17S136E, Ligand 5 RANTES, TCP228 CCL7 MCP-3, NC28, FIC, Chemokine (C-C Motif) Chemokine MARC SCYA6, Ligand 7 SCYAf CCL8 MCP-2, MCP2, Chemokine (C-C Motif) Chemokine HC14, SCYA8, Ligand 8 SCYA10 CCNA2 CCN1, CCNA, Cyclin A2 Cyclin Cyclin A CCNB1 CCNB, G/mitotic Cyclin B1 Cyclin specific cyclin B1 CCND1 BCL1, PRAD1, Cyclin D1 Cyclin G1/S-specific cyclin D1 CCND3 G/S-specific cyclin Cyclin D3 Cyclin D3, D3-type cyclin CCNE1 Cyclin E, CCNE Cyclin E1 Cyclin CCR1 CC-CKR-1, Chemokine (C-C motif) Chemokine Receptor CMKR1, MIP1aR, Receptor 1 RANTES-R, SCYAR1 CCR3 CC-CKR-3, Chemokine (C-C motif) Chemokine Receptor CMKBR3, CKR3, Receptor 3 Eotaxin receptor CCR5 CKR-5, CKR5, Chemokine (C-C motif) Chemokine Receptor chemr13, CC-CKR Receptor 5 5, CMKBR5 CD37, CD3-Zeta, CD3H, CD3 Antigen, Zeta Cell Marker CD3Q, T3Z, TCRZ. Polypeptide CD4 p55, T-cell antigen CD4 Antigen Cell Marker T4fleu3 CD8, LEU2, MAL, CD8 Antigen, Alpha Cell Marker p32, CD8 T-cell Polypeptide antigen LEU2 CD14 LPS-R, Monocyte CD14 Antigen Cell Marker differentiation antigen CD14 CD19 LEU12, B CD19 Antigen Cell Marker? Growth lymphocyte antigen Factor CD19 CD22 BL-CAM, Leu-14, CD22 Antigen Cell Marker SIGLEC2 CD34 Hematopoietic CD34 Antigen Cell Marker progenitor cell antigen, HPCA1 US 2004/0225449 A1 Nov. 11, 2004 30
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification CD44 CD44R, IN, MC56, CD44 Antigen Cell Marker/Receptor MDU2, MDU3, MIC4, Pgp1, LHR CD48 BCM1, BLAST, CD48 Antigen Cell Marker Lymphocyte antigen, MEM-102, BLAST1 CD68 Macrosialin, CD68 Antigen Cell Marker GP110, SCARD1 CD69 AIM, BL-AC/P26, CD69 Antigen (p60, Early T Receptor/Cell EA1, GP32/28, Cell Activation Antigen) Signaling and Leu-23, MLR-3 Activation CD86 B7-2 CD86 Antigen (CD28 antigen Cell signaling and ligand activation CDH1 ECAD, UVO, Arc Cadherin 1, Type 1, E Cell Adhesion 1, CDHE, LCAM, Cadherin (Epithelial) Uvomorulin CDH2 NCAD, CDHN Cadherin 2, Type 1, N Cell Adhesion Cadherin (Neuronal) CDK2 Cell division kinase Cyclin-Dependent Kinase 2 Enzyme: kinase 2, p33 PSK-J3, Cyclin-Dependent Kinase 4 Enzyme: kinase MGC14458, Cell division kinase 4 p21 wa?, CAP20, Cyclin-Dependent Kinase Tumor Suppressor? CIP1, SDI1, MDA Inhibitor 1A Cell Cycle Control 6, MDA6, PIC1 p16, MTS1, INK4, Cyclin-Dependent Kinase Tumor Suppressor? ARF, CDK4I, Inhibitor 2A Cell Cycle Control CDKN2, CMM2 CDKN2B p15, MTS2, TP15, Cyclin-Dependent Kinase Tumor Suppressor? p14-INK4b Inhibitor 2B Cell Cycle Control CHEK1 CHK1, Cell cycle CHK1 (Checkpoint, S. pombe) Enzyme: kinase checkpoint kinase Homologue CLDN14 DFNB29, Deafness, Claudin 14 Cell Adhesion autosomal recessive 29 COL1A1 Procollagen Collagen, Type 1, Alpha 1 Extracellular Matrixf Tissue Remodeling COL7A1 EBD1, EBR1, Collagen, Type VII, Alpha 1 Extracellular Matrix EBDCT CRABP2 RBP6, CRABP-II, Cellular Retinoic Acid Binding Transcriptional CRABPII Protein 2 Regulator/Small Molecule Binding Protein CREB3 LZIP, MGC15333, cAMP Responsive Element Transcription Factor MGC19782 Binding Protein 3 (Luman) CRP PTX1 C-Reactive Protein, Pentraxin Acute Phase Protein Related CSF2 Colony Stimulating Factor 2 Cytokine/Growth (Granulocyte-Monocyte) Factor CSF3 Colony Stimulating Factor 3 Cytokine/Growth (Granulocyte) Factor CTGF NOV2, IGFBP8, Connective Tissue Growth Growth Factor? Wound HCS24, CCN2, Factor Response IGFBPR2 CTLA4 CD152 Cytotoxic T-Lymphocyte Membrane Protein? Associated Protein 4 Immune Response CTNNA1 Cadherin-associated Catenin, Alpha 1 Cell Adhesion? Cell protein, CAP102 Structure CTSB APPS, CPSB, APP Cathepsin B Proteinase: thiol Secretase CTSZ, CTSX Cathepsin Z. Proteinase: thiol CX3CR1 CCRL1, CMK Chemokine (C-X3-C) Receptor 1 Chemokine Receptor BRL-1CMKDR1, GPR13, GPRV28 CXCL1 GRO1, SCYB1, Chemokine (C-X-C Motif) Chemokine/Growth GROA, MGSA, Ligand 1 Factor/Oncogene MGSA-a, NAP-3 CXCL2 GRO2, SCYB2, Chemokine (C-X-C Motif) Chemokine/Growth MIP2A, GROB, Ligand 2 Factor GROb US 2004/0225449 A1 Nov. 11, 2004 31
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification pCXCL1 2 Primate Chemokine (C-X-C Motif) Chemokine/Growth (Cynomologous Ligand 1; Chemokine (C-X-C Factor Monkey) Gene, Motif) Ligand 2 GRO1; GRO2 CXCL10 SCYB10, IP10, Chemokine (C-X-C Motif) Chemokine IFI10, CRG-2, Ligand 10 MOB-1 CXCL12 SCYB12, SDF1, Chemokine (C-X-C Motif) Chemokine PBSF, TPAR1, Ligand 12 hIRH CXCR3 GPR9, CD183, Chemokine (C-X-C Motif) Chemokine Receptor CKR-L2, IP10-R, Receptor 3 Mig-R, MigR, IP10 CXCR4 Fusin, HSY3RR, Chemokine (C-X-C Motif) Chemokine Receptor HM89, LAP3, Receptor 4 LCR1, LESTR, NPYRL CYBB NOX2, CGD, Cytochrome B-245 Beta Enzyme: redox GP91-PHOX, Polypeptide GP91 PHOX, GP91-1 CYP1A1 CYP1, AHRR, Cytochrome P450, Subfamily I Enzyme: metabolism AHH, P450DX, P1 (Aromatic Compound 450, P450-C, CP11 Inducible), Polypeptide 1 CYP1A2 CP12, P3-450, Cytochrome P450, Subfamily I Enzyme: metabolism P450(PA) (Aromatic Compound Inducible), Polypeptide 2 CYP2C19 Cytochrome P450, Subfamily Enzyme: metabolism IIC (Mephenytoin 4 Hydroxylase), Polypeptide 19 CYP2D6 Cytochrome P450, Subfamily Enzyme: metabolism IID (Debrisoquine, Sparteine, etc., -Metabolizing), Polypeptide 6 Cytochrome P450, Subfamily Enzyme: metabolism IIE (Ethanol-Inducible), Polypeptide 1 CYP3A4 P450C3, Cytochrome P450, Subfamily Enzyme: metabolism P450PCN1, CP34, IIIA (Niphedipine Oxidase), NF-25 Polypeptide 4 DAD1 DAD-1 Defender Against Cell Death 1 Enzyme? Apoptosis Inhibitor DC13* *Not a HUGO gene DC13 Protein Not Classified symbol DCN DSPG2, PG-S2, Decorin Extracellular Matrix PG40, PGII, Proteoglycan II, PGS2 DFFB CAD, CPAN, DNA Fragmentation Factor, 40 kD, Enzyme: nuclease DFF2, DFF40 Beta Polypeptide (Caspase-Activated Dnase) DPP4 CD26, ADCP2, Dipeptidylpeptidase IV Proteinase DPPIV DSG1 Desmoglein 1 Membrane protein DTR DTS, HB-EGF, Diphtheria Toxin Receptor Cell Signaling? HBEGF, HEGFL Mitogen DUSP1 CL100, PTPN10, Dual Specificity Phosphatase 1 Enzyme: phosphatase HVH1, MKP-1, VH1, MKP1 ECE1 ECE, ECE-1 Endothelin Converting Metalloproteinase Enzyme 1 EDN1 ET1 Endothelin 1 Peptide Hormone EDR2 Early Development Regulator 2 Transcription Factor EGR1 NGF1A, KROX-24, Early Growth Response 1 Transcription Factor TIS8, ZIF-268, GOS30, NGFIA, ZNF225 ELA2 Medullasin, NE, Elastase 2, Neutrophil Proteinase: serine SERP1, PMN elastase EPHX1 EPHX, MEH, EH, Epoxide Hydrolase 1, Enzyme: metabolism EPOX, FHS Microsomal (Xenobiotic) US 2004/0225449 A1 Nov. 11, 2004 32
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification ERBB2 C-erbB-2, HER2, Verb-b2 Erythroblastic Oncogene/Receptor NEU, NGL, Leukemia Viral Oncogene p185erbB2, TKR1 Homolog 2 Herstatin ERBB3 HER3, c-erbB3, Verb-b2 Erythroblastic Oncogene/Receptor Oncogene ERBB3 Leukemia Viral Oncogene Homolog 3 ESR1 ER, ER-alpha, ESR, Estrogen Receptor 1 Receptor? ESRA, Era, NR3A1 Transcription Factor Thromboplastin, Coagulation Factor III Enzyme: redox TFA, CD142, Factor II (coagulation), HTF FADD MORT1, Fas (TNFRSF6)-Associated Apoptosis?Co MGC8528, Via Death Domain Receptor Mediator of receptor-induced toxicity FAP Seprase, FAPA Fibroblast Activation Protein, Liver Health Indicatorf Alpha Subunit Proteinase FCGR1A Fc fragment of IgG, high affinity receptor BFGF, FGFB, Fibroblast Growth Factor 2 Growth Factor HBGF-2, HBGH-2, (Basic) Prostatropin KGF, Keratinocyte Fibroblast Growth Factor 7 Growth Factor Growth Factor FGF18 ZFGFS Fibroblast Growth Factor 18 Growth Factor FLT1 VEGFR1, FRT, FMS-Related Tyrosine Kinase 1 Receptor FLT CIG, FN, LETS, Fibronectin 1 Cell Adhesion LETS FNZ, FINC FOLH1 PSMA, GCP2, Folate Hydrolase (Prostate Enzyme: hydrolase FOLH, NAALAD1, Specific Membrane Antigen) 1 PSM FOS Oncogene FOS, V-fos FBJ Murine Oncogene? GOST Osteosarcoma Viral Oncogene Transcription Factor Homolog FTL Ferritin, light peptide GADD45A GADD45, DDIT1 Growth Arrest and DNA Cell Cycle Control/ Damage Inducible, Alpha Apoptosis GLP1, GLP2, Glucagon Peptide Hormone GRPP, Glicentin related polypeptide GCGR GGR, GL-R Glucagon Receptor Receptor GCK Hexokinase 4, GK, Glucokinase Enzyme: kinase GLK, HK4, HKIV, HXKP, MODY2 GCLC GCS, GLCLC, Glutamate-Cysteine Ligase, Enzyme: ligase GLCL Catalytic Subunit GFPT1 GFA, GFAT, Glutamine-Fructose-6- Enzyme: GFAT1, GFPT Phosphate Transaminase 1 amidotransferase GGT1 Glutamyl Gamma-Glutamyltransferase 1 Liver Health Indicator transpeptidase, GTG, GGT, CD224 GA1 CX43, Connexin Gap Junction Protein, Alpha 1, Channel Protein? 43, DFNB38, Heart 43 kD Transporter connexin GSR GR, GRASE, Glutathione Reductase Enzyme: redox GLUR, GRD1 GST Primers/probe set Glutathione S-Transferase Enzyme: metabolism nonspecific for all Class members of GST family GSTA1 2 GST-epsilon, Glutathione S-Transferase A1; Enzymes: metabolism GSTA1-1, GTH1; Glutathione S-Transferase A2 GST gamma, GST2 GSTM1 GST1, GSTM1-1, Glutathione S-Transferase M1 Enzyme: metabolism GTH4, GSTM1a 1a, GSTM1b-1b GSTT1 Glutathione Glutathione-S-Transferase Enzyme: metabolism transferase T1-1 Theta 1 US 2004/0225449 A1 Nov. 11, 2004 33
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification GYS1 GYS Glycogen Synthase 1 (Muscle) Enzyme: glycogen metabolism CTLA1, Granzyme Granzyme B Proteinase 2, C11, CCPI, CGL-1, CSP-B, GRB MOP1, ARNT Hypoxia-Inducible Factor 1, Transcription Factor Interacting Protein Alpha Subunit HK2 HKII, HXK2, Hexokinase 2 Enzyme: hexokinase Muscle form Hexokinase HLA-DRB1 HLA class II Major Histocompatibility Histocompatibility histocompatibility Complex, Class II, DR Beta 1 antigen, DR-1 beta chain HMGA1 HMGIY, HMG High-Mobility Group AT Transcriptional I/HMG-Y, HMG Hook 1 Regulator/Oncogene I/Y, HMG-R HMGB1 HMG1, HMG3, High-Mobility Group Box 1 Transcriptional Amphoterin Regulator HMGIY High mobility group protein, DNA binding - isoforms I and Y transcriptional regulation - oncogene HMOX1 HO1, HO, HO-1, Heme Oxygenase (Decycling) 1 Enzyme: redox bK286B10, Heat shock protein 32 K HSPA1A HSP-70, HSP70-1 Heat Shock Protein 1A, 70 kD Cell Signaling and Activation HUPAP* *Not a HUGO gene Human Prostate-Associated Proteinase symbol Proteinase ICAM1 CD54, BB2, Human Intercellular Adhesion Cell Adhesion/Matrix rhinovirus receptor Molecule 1 Protein ICOS AILIM Inducible T-Cell Co-Stimulator Receptor IF16 IFNGIP1 Interferon, Gamma-Inducible Transcriptional Protein 16 Repressor IFNA2 8 10 Interferon, Alpha 2: Interferon, Cytokines/Apoptosis Alpha 8: Interferon, Alpha 10 IFNG Interferon, Gamma Cytokine IGF1R Insulin-Like Growth Factor 1 Receptor/Enzyme: Receptor kinase IGFBP3 IBP3, IGFBP-3, Insulin-Like Growth Factor IGF-Binding Protein IBP-3, IGF-binding Binding Protein 3 protein 3 IL1A IL1a, IL1-Alpha, Interleukin 1, Alpha Cytokine IL-1A, IL1, IL1E1, Hematopoietin-1 IL1b, IL1-Beta, IL Interleukin 1, Beta Cytokine 1B, IL1F2, Catabolin IL1R1RA, IL1RA, Interleukin 1 Receptor, Type I Receptorf Cell p80, IL-1R-1, IL Signaling and 1 R-alpha, IL1R, Activation CD12 ICIL-1RA, IL1F3, Interleukin 1 Receptor Cytokine Antagonist IL-1RA, IRAP, IL Antagonist 1RN, IL1RA IL2 TCGF Interleukin 2 Cytokine/Growth Factor IL2R, P55, TCGFR, Interleukin 2 Receptor, Alpha Receptorf Cell CD25, TAC antigen Signaling and Activation IL4 BSF1 Interleukin 4 Cytokine/Growth Factor IL5 EDF, Eosinophil Interleukin 5 Cytokine/Growth differentiation Factor factor, TRF, E-CSF Interferon beta 2, Interleukin 6 Cytokine/Growth IFNB2, BSF2, HSF Factor IL7 IL-7 Interleukin 7 Cytokine/Growth Factor US 2004/0225449 A1 Nov. 11, 2004 34
TABLE 8-continued Master List for Gene Expression Panels
Symbol Alias(es) Nam e Classification ILFR IL-7R-alpha, Inter eukin 7 Receptor Receptor CD127 antigen, CDW127, p90 IL 7R IL8 CXCL8, SCYB8, Inter eukin 8 Chemokine MDNCF IL10 CSIF, IL-10, TGIF, Inter eukin 10 Cytokine Cytokine synthesis inhibitory factor IL12p40, NKSF2, Inter eukin 12 (p40) Cytokine/Growth CLMF, CLMF2, Factor p40 IL 3 IL-13, NC30, p800 eukin 3 Cytokine IL15 IL-2-like cytokine eukin 15 Cytokine/Growth Factor IL17 CTLA8, IL-17, IL Inter eukin 17 Cytokine 17A, IL17A IL18 IGIF, IL-18, IL-1 g, Inter eukin 18 Cytokine/Growth IL1R4, Interleukin Factor 1 g IL18BPa, IL18BPb, Inter eukin-18 Binding Protein Cytokine Inhibitor IL18BPC IL18R1 Inter eukin 18 receptor 1 Membrane protein INS None Insulin Hormone IRFS IRF-5 Inter eron Regulatory Factor 5 Transcription Factor IRS1 HIRS-1 Insulin Receptor Substrate 1 Signal Transduction ITGAM CD11B Integrin, alpha M; complement Integrin receptor IVL None Invo lucrin Cell Structure JUN CJUN, Protooncogene V-jun Avian Sarcoma Virus 17 Transcription Factor c-Jun, Oncogene Homolog AP-1, AP1 KAI1 SAR2, CD82, ST6, Kangai 1 Tumor Suppressor R2, IA4, 4F9, C33, GR15 K-ALPHA *Not a HUGO gene Tubulin, Alpha, Ubiquitous Cell Structure 1. : symbol KITLG MGF (formerly), KIT Ligand Liver Health Indicatorf Stem cell factor Growth Factor (SCF), SF, KL-1, KTL KLK1 Tissue kallikrein, Kallikrein 1 (Renal/Pancreas/ Proteinase KLKR, Kallikrein Salivary) serine protease 1 hGK-1 Glandular Kallikrein 2 (Prostatic) Proteinase kallikrein PSA, APS, KLKB1, Kallikrein 3 (Prostate Specific Proteinase Semenogelase, Antigen) Seminin KRTS EBS2, CK5, K5, Keratiin 5 Cell Structure Cytokeratin 5 KRT8 K8, CK8, CARD2, Keratiin 8 Cell Structure CYK8, Cytokeratin 8 KRT14 EBS3, EBS4, Kera in 14 Cell Structure CK14, K14, Cytokeratin 14 KRT16 K16, NEPPK, Kera in 16 Cell Structure CK16, Cytokeratin 16 KRT19 K19, Keratin (type Kera in 19 Cell Structure I) 40-kD Lipo polysaccharide binding Membrane protein protein LGALS3 LGALS2, Galectin Lectin, Galactoside-Binding, Liver Health Indicator 3, GALBP, MAC2 Solu ble 3 LGALS8 PCTA-1, Galectin Lectin, Galactoside-Binding, Cell Adhesion/Growth 8, PCTA1, Poé6 Solu ble 8 and Differentiation CBP LTA TNFSF1, Tumor Lym photoxin, Alpha Cytokine necrosis factor beta (formerly), TNFB US 2004/0225449 A1 Nov. 11, 2004 35
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification MADD DENN, IG20, MAP-Kinase Activating Death Co-Receptor Insulinoma Domain glucagonoma protein 20 MAP3K1 MAPKKK1, Mitogen-Activated Protein Enzyme: kinase MEKK1, MEKK, Kinase Kinase Kinase 1 MAP/ERK kinase kinase 1 MAP3K14 NF-kB Inducing Mitogen-Activated Protein Enzyme: kinase Kinase, NIK, Kinase Kinase Kinase 14 HSNIK, FTDCR1B, HS MAPK1 ERK2, ERK, ERT1, Mitogen-Activated Protein Enzyme: kinase MAPK2, PRKM1, Kinase 1 p38, p40, p41 MAPK8 JNK1, JNK, Mitogen-Activated Protein Enzyme: kinase SAPK1, PRKM8, Kinase 8 JNK1A2, JNK21B1/2 MAPK14 CSBP, CSBP1, p38, Mitogen-Activated Protein Enzyme: kinase Mxi2, PRKM14, Kinase 14 PRKM15 MDM2 HDM2, MDM2, Transformed 3T3 Cell Transcription Factor/ Oncoprotein Double Minute 2, p53 Binding Oncogene MDM2, p53 Protein (Mouse) binding protein MDM2 MHC2TA C2TA, CIITA MHC Class II Transactivator DNA-Binding Protein? Activator MIF Macrophage migration Cell signaling and inhibitory factor growth Iactor MMP1 Collagenase, CLG, Matrix Metalloproteinase 1 Metalloproteinase CLGN, Fibroblast collagenase MMP2 Gelatinase, CLG4A, Matrix Metalloproteinase 2 Metalloproteinase CLG4, TBE-1, Gelatinase A MMP3 Stromelysin, Matrix Metalloproteinase 3 Metalloproteinase STMY1, STMY, SL-1, STR1, Transin-1 MMP8 Neutrophil Matrix Metalloproteinase 8 Metalloproteinase collagenase, CLG1, HNC, PMNL-CL MMP9 Gelatinase B, Matrix Metalloproteinase 9 Metalloproteinase CLG4B, GELB, Macrophage gelatinase MMP12 Macrophage Matrix Metalloproteinase 12 Metalloproteinase elastase, HME, MME MMP13 Collagenase 3, Matrix Metalloproteinase 13 Metalloproteinase CLG3 MMP15 MT2-MMP, MMP Matrix Metalloproteinase 15 Metalloproteinase 15, SMCP-2, (Membrane-Inserted) MT2MMP, MTMMP2 MMP19 MMP18 (formerly), Matrix Metalloproteinase 19 Metalloproteinase RASI-1, RASI MNDA None Myeloid Cell Nuclear Cell Signaling and Differentiation Antigen Activation MP1 Metalloprotease 1 Proteinase/Proteinase Inhibitor MPO E.C. 1111.7 Myeloperoxidase Enzyme: redox MRE11A MRE11, ATLD, Meiotic Recombination (S. Enzyme: nuclease HNGS1, MRE11B cerevisiae) 11 Homolog A MYC c-MYC, Oncogene V-myc Avian Transcription Factor/ MYC Myelocytomatosis Viral Oncogene Oncogene Homolog US 2004/0225449 A1 Nov. 11, 2004 36
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification D8S1992, N33 Putative Prostate Cancer Tumor Suppressor protein, *Not a Tumor Suppressor HUGO gene symbol NFKB1 KBF1, EBP-1, Nuclear Factor of Kappa Light Transcription Factor NFKB p50 Polypeptide Gene Enhancer in B-Cells 1 (p105) NFKBIB TRIP9, IKBB, Nuclear Factor of Kappa Light Transcriptional Thyroid hormone Polypeptide Gene Enhancer in Regulator receptor interactor 9 B-Cells Inhibitor, Beta NOS1 NOS, N-NOS, Nitric Oxide Synthase 1 Enzyme: redox NNOS, Neuronal (Neuronal) NOS, Constitutive NOS NOS2A iNOS, NOS2 Nitric Oxide Synthase 2A Enzyme: redox (Inducible) NOS3 eNOS, cNOS, Nitric Oxide Synthase 3 Enzyme: redox ECNOS (Endothelial) NR12 PXR, PAR2, Nuclear Receptor Subfamily 1, Receptor? Pregnane X Group I, Family 2 Transcription Factor Receptor, PARq, PRR, SXR NR13 Constitutive Nuclear Receptor Subfamily 1, Receptor? androstane receptor Group I, Family 3 Transcription Factor beta, CAR, MB67 NRP1 NRP, VEGF165R Neuropilin 1 Receptor ORM1 A1AGP, Alpha 1 Orosomucoid 1 Liver Health Indicator acid glycoprotein, AGP-A, ORM OXCT OXCT1, SCOT 3-Oxoacid CoA Transferase Enzyme: transferase PART1* DKFZP586D0823; Prostate Androgen-Regulated Unclassified *Not a HUGO gene Transcript 1 symbol PCA3 Prostate-specific Prostate Cancer Antigen 3 Unclassified gene DD3, DD3 PCANAP7 IPCA7, IPCA-7 Prostate Cancer Associated Unclassified Protein 7 PCGEM1* *Not a HUGO gene Prostate-Specific Non-Coding Riboregulator symbol Gene PCK1 PEPCK-C, Phosphoenolpyruvate Enzyme: lyase PEPCK1, Cytosolic Carboxykinase 1 PEPCK PCNA DNA polymerase Proliferating Cell Nuclear DNA-Binding Protein delta auxiliary Antigen protein PCTK1 PCTAIRE1, PCTAIRE Protein Kinase 1 Enzyme: kinase PCTGAIRE PDCD8 AIF, Apoptosis Programmed Cell Death 8 Enzyme: redox Inducing Factor PDEF: *Not a HUGO gene Prostate Epithelium Specific Transcription Factor symbol Ets Transcription Factor CXCL4, SCYB4 Platelet Factor 4 (Chemokine Chemokine (C-X-C Motif) Ligand 4) PHC2 EDR2, Early Polyhomeotic-Like 2 Inhibitor or Repressor development (Drosophila) regulator 2, HPH2 SKALP, ELAFIN, Proteinase Inhibitor 3 (Skin Proteinase Inhibitor ESI, Elastase Derived) specific inhibitor GRB1, PI3K, Phosphoinositide-3-Kinase, Regulatory Adaptor PI3KA, Ptdins-3- Regulatory Subunit, Protein kinase p85-alpha Polypeptide 1 PITRM1 MP1, hMP1, Pitrilysin Metalloproteinase 1 Proteinase KIAA1104 PLA2B, PLA2L, Phospholipase A2, Group IIA Enzyme: phospholipase MOM1, RASFA, NPS-PLA2 PLA2G7 PAFAH, LDL Phospholipase A2, Group VII Enzyme: hydrolase PLA2 PLAT TPA, T-PA, Plasminogen Activator, Tissue Proteinase Alteplase, Reteplase US 2004/0225449 A1 Nov. 11, 2004 37
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification PLAU UPA, URK, Plasminogen Activator, Proteinase Plasminogen Urokinase activator (urinary) PLAUR UPAR, CD87, Plasminogen Activator, Receptor URKR Urokinase Receptor PNKP PNK, DNA kinase Polynucleotide Kinase 3'- Enzyme: phosphatase Phosphatase POV1 ROO504, PB39 Prostate Cancer Over Unclassified expressed Gene 1 PPARA PPAR, HPPAR, Peroxisome Proliferator Receptor? NR1C1 Activated Receptor, Alpha Transcription Factor PPARG HUMPPARG, Peroxisome Proliferator Receptor? NR1C3, PPAR-g, Activated Receptor, Gamma Transcription Factor PPARG3, PPARG2, PPARG1 PRF1 Preforming protein, Perforin1 Channel Protein? HPLH2, P1, PFP, Apoptosis Pore forming protein PRKCB1 PKCB protein kinase C, beta 1 Enzyme: phosphorylase PRTN3 PR3, C-ANCA, Proteinase 3 Proteinase ACPA, MBN, AGP7, MBT, Myeloblastin, p29 PSCA None Prostate Stem Cell Antigen Cell Marker/Antigen PTEN Mutated in multiple Phosphatase and Tensin Enzyme?Tumor advanced cancers 1, Homolog Suppressor BZS, MHAM, TEPI PTGIS PGIS, PTGI, CYP8, Prostaglandin I2 (Prostacyclin) Enzyme: isomerase CYP8A1 Synthase PTGS1 COX1, COX-1, Prostaglandin-Endoperoxide Enzyme: PGG/HS, PGHS1, Synthase 1 peroxidasefdioxygenase PTGHS PTGS2 COX2, COX-2, Prostaglandin-Endoperoxide Enzyme: PGG/HS, PGHS-2, Synthase 2 peroxidasefdioxygenase PHS-2, hCox-2 PTPRC CD45, GP180, Protein Tyrosine Phosphatase Cell Marker/Receptor LCA, B220, LY5, Receptor, Type C T2OO PTX3 Pentaxin 3, TSG14, Pentaxin Related Gene, Acute Phase Protein Pentraxin 3 Rapidly Induced by IL-1b RAD52 DNA repair protein RAD52 (S. cerevisiae) DNA-Binding Protein RAD52 Homolog RB1 OSRC, PP110, Retinoblastoma 1 (Including Tumor Suppressor p105-Rb, Rb Osteosarcoma) RXRA Retinoic acid Retinoid X Receptor, Alpha Receptor? receptor RXR-a, Transcription Factor NR2B1 S100A7 PSOR1, Psoriasin 1 S100 Calcium-Binding Protein 7 Cell Cycle Control/ Small Molecule Binding Protein SAA1 2 SAA, Serum Serum Amyloid A1; Serum Acute Phase Proteins amyloid A. Amyloid A2 Amyloid A, Serum, 2 SCYA2 MCP1 Small inducible cytokine A2 Cytokine/Chemokine SCYA3 MIP alpha small inducible cytokine A3 Chemokine (MIP1a) SCYAS Rantes small inducible cytokine A5 Chemokine (RANTES) SCYB10 IFN inducible IP10 small inducible cytokine Chemokine subfamily B (Cys-X-Cys), member 10 SDF1 TPAR1 stromal cell-derived factor 1 Chemokine SELE ELAM, CD62E, Selectin E Cell Adhesion ELAM1, ESEL, LECAM2 SELL CD62L, HLHRC, Selectin L Cell Adhesion LAM1, LECAM1, LEU8 SELP CD62P, GMP-140, Selectin P Cell Adhesion GMRP, GRMP, LECAM3, PSEL US 2004/0225449 A1 Nov. 11, 2004 38
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification SERPINA1 Alpha 1 Anti Serine (or Cysteine) Proteinase Proteinase Inhibitor proteinase, AAT, Inhibitor, Clade A, Member 1 PI1, PI, A1AT SERPINA3 AACT, ACT, Serine (or Cysteine) Proteinase Proteinase Inhibitor Alpha-1-Anti Inhibitor, Clade A, Member 3 chymotrypsin SERPINB2 PAI, PAI-2, PAI2, Serine (or Cysteine) Proteinase Proteinase Inhibitor PLANH2, Inhibitor, Clade B Urokinase inhibitor (Ovalbumin), Member 2 SERPINB5 Maspin, PI5 Serine (or Cysteine) Proteinase Proteinase Inhibitorf Inhibitor, Clade B Tumor Suppressor (Ovalbumin), Member 5 SERPINC1 Antithrombin III, Serine (or Cysteine) Proteinase Proteinase Inhibitor AT3, ATIII Inhibitor, Clade C (Antithrombin), Member 1 SERPINE1 PAI1, Plasminogen Serine (or Cysteine) Proteinase Proteinase Inhibitor activator inhibitor Inhibitor, Clade E type 1, PAIE, (Ovalbumin), Member 1 PLANH1 SERPING1 C-1 esterase Serine (or Cysteine) Proteinase Proteinase Inhibitor inhibitor, C1NH, Inhibitor, Glade G (C1 C1-INH, C1L, Inhibitor), Member 1 HAE1, HAE2 (Angioedema, Hereditary) SFTPD PSPD Surfactant, pulmonary Extracellular associated protein D Lipoprotein GLUT4, Glucose Solute Carrier Family 2 Glucose Transporter transporter 4 (Facilitated Glucose Transporter), Member 4 SLC35A2 UGT, UGAT, Solute Carrier Family 35 Enzyme: metabolism UGALT, UGT1, (UDP-Galactose Transporter), UGT2, UGTL Member 2 SMAC* *Not a HUGO gene Second Mitochondria-Derived Antagonist/Apoptosis symbol, DIABLO, Activator of Caspase DIABLO-S SOCS3 CIS3, SSI3, SSI-3, Suppressor of Cytokine Inhibitor or Repressor Cish3, SOCS-3 Signaling 3 SOD2 IPO-B, MnSOD, Superoxide Dismutase 2 Enzyme: redox Indophenoloxidase B (Mitochondrial) SRP19 Signal recognition Signal Recognition Particle RNA-Binding Protein particle, ribosomal 19 kDa STAT1 STAT91 Signal Transducer and Transcription Factor Activator of Transcription 1 STAT3 APRF Signal Transducer and Transcription Factor Activator of Transcription 3 STATSA B MGF, STAT5, Signal Transducer and Transcription Factors Mammary gland Activator of Transcription 5A; factor; STAT5 Signal Transducer and Activator of Transcription 5B STEAP PRSS24, STEAP1, Six Transmembrane Epithelial Transporter/Tumor MGC19484 Antigen of the Prostate Antigen TBP TATA-Box Binding Protein Transcription Factor
TEK TIE2 tyrosine kinase, endothelial Transferase Receptor TERT TCS1, EST2, TP2, Telomerase Reverse Enzyme: reverse TRT, Hest2, Transcriptase transcriptase hTERT TGFA ETGF, TGF-alpha, Transforming Growth Factor, Cytokine/Growth EGF-like TGF, Alpha Factor?Transferase TGF type 1 DPD1, CED, Transforming Growth Factor, Cytokine/Growth HGNC:2997, TGF Beta 1 Factor beta, TGFB, TGF-b TGF-b3 Transforming Growth Factor, Cytokine/Growth Beta 3 Factor/Cell Signaling Transforming growth factor, Membrane protein beta receptor II TIE JTK14, TIE1 Tyrosine Kinase with Receptor/Signal Immunoglobulin and Transduction Epidermal Growth Factor Homology Domains US 2004/0225449 A1 Nov. 11, 2004 39
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification TIMP1 TIMP, Erythroid Tissue Inhibitor of Matrix Proteinase Inhibitorf potentiating Metalloproteinase 1 Growth Factor activity, CLGI, EPA, EPO, HCI TIMP3 SFD, HSMRK222, Tissue Inhibitor of Matrix Proteinase Inhibitor K222TA2 Metalloproteinase 3 TLR2 TIL4, Toll? Toll-Like Receptor 2 Receptorf Cell Interleukin 1 Signaling and receptor-like 4 Activation hToll, TOLL Toll-Like Receptor 4 Receptorf Cell Signaling and Activation HOX11 L2, RNX, T-Cell Leukemia, Homeobox 3 Transcription Factor Homeo box 11-like 2 TNF TNF-alpha, TNFa. Tumor Necrosis Factor, Cytokine/Growth cachectin, DIF, Member 2 TNFA, TNFSF2 FPF, TNF-R, TNF Tumor Necrosis Factor Receptor?Ce R1, TNFAR, Receptor Superfamily, Signaling and TNFR1, TNFR60, Member 1A Activation p55, p55-R TNFR2, p75, Tumor Necrosis Factor Receptor?Ce CD12Ob Receptor Superfamily, Signaling and Member 1B Activation TNFRSF11A RANK, Activator of Tumor Necrosis Factor Receptor?Ce NF-kB, ODFR, Receptor Superfamily, Signaling and PDB2 Member 11A Activation TNFRSF13B TACI, Tumor Necrosis Factor Receptor?Ce Transmembrane Receptor Superfamily, Signaling and Activator & CAML Member 13B Activation Interactor TNFRSF17 BCMA, BCM, B Tumor Necrosis Factor Receptor?Ce cell maturation Receptor Superfamily, Signaling and factor Member 17 Activation TNFRSF25 TNFRSF12 Tumor Necrosis Factor Receptor?Ce (formerly), LARD, Receptor Superfamily, Signaling and TRAMP, WSL-1, Member 25 Activation TR3, DR3 CD4OL, TRAP, Tumor Necrosis Factor Cytokine gp39, HIGM1, (Ligand) Superfamily, Member 5 CD154, IMD3, IGM, T-BAM FasL, APT1LG1, Tumor Necrosis Factor Cytokine? Apoptosis CD95L, APTL, (Ligand) Superfamily, Member 6 APO-1 TNFSF12 TWEAK, APO3L, Tumor Necrosis Factor Cytokine? Apoptosis DR3LG (Ligand) Superfamily, Member 12 TALL-1, BLYS, Tumor Necrosis Factor Cytokine/Growth TALL1, BAFF, (Ligand) Superfamily, Member Factor THANK, TNFSF20 13B TOSO* *Not a HUGO gene Regulator of Fas-Induced Apoptosis Inhibitor symbol Apoptosis p53, TRP53 Tumor Protein 53 (Li Transcription Factor/ Fraumeni Syndrome) Tumor Suppressor TRADD Tumor necrosis TNFRSF1A-Associated Via Regulatory Adaptor factor receptor-1- Death Domain Protein/Apoptosis associated protein TRAF1 EBI6, MGC:10353, TNF Receptor-Associated Regulatory Adaptor Epstein-barr virus Factor 1 Protein induced mRNA 6 TRAF2 TNF-receptor TNF Receptor-Associated Regulatory Adaptor associated protein, Factor 2 Protein MGC:45O12, TRAP3 TRAF3 CD4OBP, LAP1, TNF Receptor-Associated Signal Transduction? CAP1, CRAF1, Factor 3 Apoptosis LMP1 TREM1 TREM-1 Triggering Receptor Expressed Receptor/Cell on Myeloid Cells 1 Signaling and Activation US 2004/0225449 A1 Nov. 11, 2004 40
TABLE 8-continued Master List for Gene Expression Panels Symbol Alias(es) Name Classification Thioredoxin Reductase 1 Enzyme: redox UCP2 SLC25A8, UCPH Uncoupling Protein 2 Liver Health Indicator (Mitochondrial, Proton Carrier) UGT UDP-Glucuronosyltransferase Enzyme, metabolism VCAM1 L1CAM, CD106, Vascular Cell Adhesion Cell Adhesion/Signal INCAM-1OO Molecule 1 Transduction VDAC1 PORIN, PORIN-31 Voltage-Dependent Anion Channel Protein? HL, Plasmallemmal Channel 1 Transporter porin VEGF VPF, VEGF-A, Vascular Endothelial Growth Cytokine/Growth VEGFA, Factor Factor Vasculotropin Von Willebrand Factor Cell Adhesion? Coagulation Factor XRCCS CTC85, CTCBF, X-ray Repair Complementing Enzyme: helicase G22P2, Ku antigen Defective Repair in Chinese (80 kDa) Hamster Cells 5
We claim: of variation for repeated measurements for each constituent 1. A method for determining a profile data Set for a from the same Sample is less than 1 percent. Subject, based on a Sample from the Subject, the Sample 4. A method according to claim 2, wherein inter-assay providing a Source of RNAS, the method comprising: variability is reproducible Such that the average coefficient quantitatively measuring an amount of RNA correspond of variation for measurements for each constituent in distinct ing to each of at least two constituents from any one of Samples of the same material is less than 2 percent. Tables 1 through 8, where the profile data Set comprises 5. A method according to claim 1, wherein the profile data the measure of each constituent and wherein Such Set is informative about a biological condition for the Subject measure for each constituent is obtained under mea with respect to the circumstances of the Subject at the time Surement conditions that are Substantially reproducible. the Sample was obtained. 2. A method according to claim 1, wherein the measure 6. A method according to claim 5, wherein the profile data ment conditions that are reproducible include intra-assay Set provides guidance for medical or Surgical intervention by variability and inter-assay variability. allopathic or naturpathic means. 3. A method according to claim 2, wherein intra-assay variability is reproducible Such that the average coefficient